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1.
Automated Label-free Quantification of Metabolites from Liquid Chromatography–Mass Spectrometry Data
Erhan Kenar Holger Franken Sara Forcisi Kilian W?rmann Hans-Ulrich H?ring Rainer Lehmann Philippe Schmitt-Kopplin Andreas Zell Oliver Kohlbacher 《Molecular & cellular proteomics : MCP》2014,13(1):348-359
2.
Gregory J. Tanner Michelle L. Colgrave Malcolm J. Blundell Hareshwar P. Goswami Crispin A. Howitt 《PloS one》2013,8(2)
Background
Subjects suffering from coeliac disease, gluten allergy/intolerance must adopt a lifelong avoidance of gluten. Beer contains trace levels of hordeins (gluten) which are too high to be safely consumed by most coeliacs. Accurate measurement of trace hordeins by ELISA is problematic.Methods
We have compared hordein levels in sixty beers, by sandwich ELISA, with the level determined using multiple reaction monitoring mass spectrometry (MRM-MS).Results
Hordein levels measured by ELISA varied by four orders of magnitude, from zero (for known gluten-free beers) to 47,000 µg/mL (ppm; for a wheat-based beer). Half the commercial gluten-free beers were free of hordein by MS and ELISA. Two gluten-free and two low-gluten beers had zero ELISA readings, but contained significant hordein levels (p<0.05), or near average (60–140%) hordein levels, by MS, respectively. Six beers gave false negatives, with zero ELISA readings but near average hordein content by MS. Approximately 20% of commercial beers had ELISA readings less than 1 ppm, but a near average hordein content by MS. Several barley beers also contained undeclared wheat proteins.Conclusions
ELISA results did not correlate with the relative content of hordein peptides determined by MS, with all barley based beers containing hordein. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes; this may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique, enabling the quantification of individual hordein isoforms. This outlines the problem of relying solely on ELISA determination of gluten in beverages such as beer and highlights the need for the development of new sensitive and selective quantitative assay such as MS. 相似文献3.
Lennart Martens Matthew Chambers Marc Sturm Darren Kessner Fredrik Levander Jim Shofstahl Wilfred H. Tang Andreas R?mpp Steffen Neumann Angel D. Pizarro Luisa Montecchi-Palazzi Natalie Tasman Mike Coleman Florian Reisinger Puneet Souda Henning Hermjakob Pierre-Alain Binz Eric W. Deutsch 《Molecular & cellular proteomics : MCP》2011,10(1):R110.000133
4.
Sheng Pan Ru Chen Ruedi Aebersold Teresa A. Brentnall 《Molecular & cellular proteomics : MCP》2011,10(1):R110.003251
Glycosylation is one of the most important and common forms of protein post-translational modification that is involved in many physiological functions and biological pathways. Altered glycosylation has been associated with a variety of diseases, including cancer, inflammatory and degenerative diseases. Glycoproteins are becoming important targets for the development of biomarkers for disease diagnosis, prognosis, and therapeutic response to drugs. The emerging technology of glycoproteomics, which focuses on glycoproteome analysis, is increasingly becoming an important tool for biomarker discovery. An in-depth, comprehensive identification of aberrant glycoproteins, and further, quantitative detection of specific glycosylation abnormalities in a complex environment require a concerted approach drawing from a variety of techniques. This report provides an overview of the recent advances in mass spectrometry based glycoproteomic methods and technology, in the context of biomarker discovery and clinical application.With recent advances in proteomics, analytical and computational technologies, glycoproteomics—the global analysis of glycoproteins—is rapidly emerging as a subfield of proteomics with high biological and clinical relevance. Glycoproteomics integrates glycoprotein enrichment and proteomics technologies to support the systematic identification and quantification of glycoproteins in a complex sample. The recent development of these techniques has stimulated great interest in applying the technology in clinical translational studies, in particular, protein biomarker research.While glycomics is the study of glycome (repertoire of glycans), glycoproteomics focuses on studying the profile of glycosylated proteins, i.e. the glycoproteome, in a biological system. Considerable work has been done to characterize the sequences and primary structure of the glycan moieties attached to proteins (1–3), and their structural alterations related to cancer (4–6). Recent reports have provided a comprehensive overview of the concept of glycomics and its prospective in biomarker research (7–10). In contrast, this review is focused on recent developments in glycoproteomic techniques and their unique application and technical challenge to biomarker discovery.
Open in a separate windowProtein biomarker development is a complex and challenging task. The criteria and approach applied for developing each individual biomarker can vary, depending on the purpose of the biomarker and the performance requirement for its clinical application (16, 17). In general, it has been suggested that the preclinical exploratory phase of protein biomarker development can be technically defined into four stages (18), including initial discovery of differential proteins; testing and selection of qualified candidates; verification of a subset of candidates; assay development and pre-clinical validation of potential biomarkers. Thanks to recent technological advances, mass spectrometry based glycoproteomics is now playing a major role in the initial phase of discovering aberrant glycoproteins associated with a disease. Glycoprotein enrichment techniques, coupled with multidimensional chromatographic separation and high-resolution mass spectrometry have greatly enhanced the analytical dynamic range and limit of detection for glycoprotein profiling in complex samples such as plasma, serum, other bodily fluids, or tissue. In addition, candidate-based quantitative glycoproteomics platforms have been introduced recently, allowing targeted detection of glycoprotein candidates in complex samples in a multiplexed fashion, providing a complementary tool for glycoprotein biomarker verification in addition to antibody based approaches. It is clear that glycoproteomics is gaining momentum in biomarker research.
Glycoproteomics in Biomarker Discovery and Clinical Study
Most secretory and membrane-bound proteins produced by mammalian cells contain covalently linked glycans with diverse structures (2). The glycosylation form of a glycoprotein is highly specific at each glycosylation site and generally stable for a given cell type and physiological state. However, the glycosylation form of a protein can be altered significantly because of changes in cellular pathways and processes resulting from diseases, such as cancer, inflammation, and neurodegeneration. Such disease-associated alterations in glycoproteins can happen in one or both of two ways: 1) protein glycosylation sites are either hypo, hyper, or newly glycosylated and/or; 2) the glycosylation form of the attached carbohydrate moiety is altered. In fact, altered glycosylation patterns have long been recognized as hallmarks in cancer progression, in which tumor-specific glycoproteins are actively involved in neoplastic progression and metastasis (5, 6, 11, 12). Sensitive detection of such disease-associated glycosylation changes and abnormalities can provide a unique avenue to develop glycoprotein biomarkers for diagnosis and prognosis. In addition, intervention in the glycosylation and carbohydrate-dependent cellular pathways represent a potential new modality for cancer therapies (6, 11, 13). 14, 15) that are glycosylated proteins or protein complexes.Table I
Listing of some of the US Food and Drug Administration (FDA) approved cancer biomarkers| Protein target | Glycosylation | Detection | Source | Disease | Clinical biomarker |
|---|---|---|---|---|---|
| α-Fetoprotein | Yes | Glycoprotein | Serum | Nonseminomatous testicular cancer | Diagnosis |
| Human chorionic gonadotropin-β | Yes | Glycoprotein | Serum | Testicular cancer | Diagnosis |
| CA19–9 | Yes | Carbohydrate | Serum | Pancreatic cancer | Monitoring |
| CA125 | Yes | Glycoprotein | Serum | Ovarian cancer | Monitoring |
| CEA (carcinoembryonic antigen) | Yes | Protein | Serum | Colon cancer | Monitoring |
| Epidermal growth factor receptor | Yes | Protein | Tissue | Colon cancer | Therapy selection |
| KIT | Yes | Protein (IHC) | Tissue | Gastrointestinal (GIST) cancer | Diagnosis/Therapy selection |
| Thyroglobulin | Yes | Protein | Serum | Thyroid cancer | Monitoring |
| PSA-prostate-specific antigen (Kallikrein 3) | Yes | Protein | Serum | Prostate cancer | Screening/Monitoring/Diagnosis |
| CA15–3 | Yes | Glycoprotein | Serum | Breast cancer | Monitoring |
| CA27–29 | Yes | Glycoprotein | Serum | Breast cancer | Monitoring |
| HER2/NEU | Yes | Protein (IHC), Protein | Tissue, Serum | Breast cancer | Prognosis/Therapy selection/Monitoring |
| Fibrin/FDP-fibrin degradation protein | Yes | Protein | Urine | Bladder cancer | Monitoring |
| BTA-bladder tumour-associated antigen (Complement factor H related protein) | Yes | Protein | Urine | Bladder cancer | Monitoring |
| CEA and mucin (high molecular weight) | Yes | Protein (Immunofluorescence) | Urine | Bladder cancer | Monitoring |
Glycoproteomics Approaches
Glycoproteomic analysis is complicated not only by the variety of carbohydrates, but also by the complex linkage of the glycan to the protein. Glycosylation can occur at several different amino acid residues in the protein sequence. The most common and widely studied forms are N-linked and O-linked glycosylation. O-linked glycans are linked to the hydroxyl group on serine or threonine residues. N-linked glycans are attached to the amide group of asparagine residues in a consensus Asn-X-Ser/Thr sequence (X can be any amino acid except proline) (19). Other known, but less well studied forms of glycosylation include glycosylphosphatidylinositol anchors attached to protein carboxyl terminus, C-glycosylation that occurs on tryptophan residues (20), and S-linked glycosylation through a sulfur atom on cysteine or methionine (21, 22). Our following discussion is focused on glycoproteomic analysis of the most common N-linked and O-linked glycoproteins.A comprehensive analysis of glycoproteins in a complex biological sample requires a concerted approach. Although the specific methods for sample preparation can be different for different types of samples (e.g. plasma, serum, tissue, and cell lysate), a glycoproteomics pipeline typically consists of glycoprotein or glycopeptide enrichment, multidimensional protein or peptide separation, tandem mass spectrometric analysis, and bioinformatic data interpretation. For glycoprotein-based enrichment methods, proteolytic digestion can be performed before or after glycan cleavage, depending on the specific workflow and enrichment methods used. For glycopeptide enrichment, proteolytic digestion is typically performed before the isolation step so that glycopeptides, instead of glycoproteins, can be captured. For quantitative glycoproteomics profiling, additional steps, such as differential stable isotope labeling of the sample and controls, are required. Fig. 1 illustrates the general strategy for an integrated glycoproteomics analysis.Open in a separate windowFig. 1.The strategies of mass spectrometry based glycoproteomic analysis.Glycoproteins or glycopeptides can be effectively enriched using a variety of techniques (see below). Following the enrichment step, the workflow then splits into two directions: glycan analysis and glycoprotein analysis. The strategies for glycan analysis have been discussed in several reviews and will not be covered in this report. For glycoprotein analysis, bottom-up workflows (“shotgun proteomics”—peptide based proteomics analysis) (23) are still most common, providing not only detailed information of a glycoprotein profile, but also the specific mapping of glycosylation sites. It is notable that the reliable analysis of mass spectrometric data in glycoproteomic studies largely relies on bioinformatic tools and glyco-related databases that are available. An increasing number of algorithms and databases for glycan analysis have been developed and well documented in several recent reviews (24–26). For glycoprotein and glycopeptide sequence analysis, a large number of well-characterized and annotated glycoproteins can be found in the UniProt Knowledgebase. In addition, many glycopeptide mass spectra are now available in the continually expanding PeptideAtlas library (27), which stores millions of high-resolution peptide fragment ion mass spectra acquired from a variety of biological and clinical samples for peptide and protein identification. Ultimately, all the data obtained from different aspects of the workflow need to be merged and interpreted in an integrated fashion so that the full extent of glycosylation changes associated with a particular biological state can be better revealed. To the best of our knowledge, the complete glycoform analysis of any glycoprotein in a specific cell type under any specific condition has not yet been accomplished for any glycoprotein with multiple glycosylation sites. Current technology can define the glycan compliment and profile the glycoproteins, but is not capable of putting them together to define the molecular species present. To date, such integrated studies still remain highly challenging, even with advanced tandem mass spectrometry technologies and growing bioinformatic resources (26, 28–31).Enrichment of the Glycoproteome
Characterization of the glycoproteome in a complex biological sample such as plasma, serum, or tissue, is analytically challenging because of the enormous complexity of protein and glycan constituents and the vast dynamic range of protein concentration in the sample. The selective enrichment of the glycoproteome is one of the most efficient ways to simplify the enormous complexity of a biological sample to achieve an in-depth glycoprotein analysis. Two approaches for glycoprotein enrichment have been widely applied: lectin affinity based enrichment methods (31–36) and hydrazide chemistry-based solid phase extraction methods (37–42). Recent studies have demonstrated that the two methods are complementary and a very effective means for the enrichment of glycoproteins or glycopeptides from human plasma and other bodily fluids (38, 39, 43). In addition, glycoprotein and glycopeptide enrichment using boronic acid (44, 45), size-exclusion chromatography (46), hydrophilic interaction (47) and a graphite powder microcolumn (48) have been reported.Lectin affinity enrichment is based on the specific binding interaction between a lectin and a distinct glycan structure attached on a glycoprotein (49, 50). There are a variety of lectin species that can selectively bind to different oligosaccharide epitopes. For instance, concanavalin A (ConA) binds to mannosyl and glucosyl residues of glycoproteins (51); wheat germ agglutinin (WGA) binds to N-acetyl-glucosamine and sialic acid (52); and jacalin (JAC) specifically recognizes galactosyl (β-1,3) N acetylgalactosamine and O-linked glycoproteins (53). Lectin affinity enrichment has been designed to enrich glycoproteins with specific glycan attachment from plasma, serum, tissue, and other biological samples through affinity chromatography and other methods. Multiple lectin species can also be combined to isolate multiple types of glycoproteins in complex biological samples (54–59). Concanavalin A and wheat germ agglutinin, as well as jacalin are often used together to achieve a more extensive glycproteome characterization (31, 34, 57, 59, 60). Several reports have demonstrated a multilectin column approach to achieve a global enrichment of glycoproteins with various glycan attachments from serum and plasma (31, 34, 59, 61, 62). A recent study has developed a “filter aided sample preparation (FASP)” based method, which allows highly efficient enrichment of glycopeptides using multi-lectins (63). To date, most of the work using lectin affinity for targeted glycoprotein enrichment has focused on N-glycosylation because the binding specificity of lectin for O-glycosylation is less satisfactory. To overcome such caveat, efforts have been made using serial lectin columns of concanavalin A and jacelin in tandem to isolate O-glycopeptides from human serum (35).A hydrazide chemistry-based method has been applied to isolate glycoproteins and glycopeptides through the formation of covalent bonding between the glycans and the hydrazide groups (37). The carbohydrates on glycoproteins are first oxidized to form aldehyde groups, which sequentially react with hydrazide groups that are immobilized on a solid surface. The chemical reaction conjugates the glycoproteins to the solid phase by forming the covalent hydrazone bond. Although, conceptually, the majority of the glycoproteins in a biological sample can be captured using this method, the further analysis of the captured glycoproteins is practically limited by the method that can cleave glycoproteins or glycopeptides from the solid phase. Because there is a lack of efficient enzymes or chemicals that can specifically deglycosylate and/or release O-linked glycoproteins or glycopeptides from the solid phase, most of the studies have applied this method solely for N-linked glycoprotein analysis. PNGase F is the enzyme that can specifically release an N-glycosylated proteins or peptides (except those carrying α1→3 linked core fucose (38)) from its corresponding oligosaccharide groups. The hydrazide chemistry method is not only highly efficient in enriching N-linked glycoproteins or glycopeptides from a complex environment, but also allows great flexibility in its applications, such as capturing extracellular N-glycoproteins on live cells to monitor their abundant changes because of cell activation, differentiation, or other cellular activities (64). This method can be readily automated for analyzing a large quantity of samples.Recent studies have compared glycoprotein isolation methods. One study assessed lectin-based protocols and hydrophilic interaction chromatography for their performance in enriching glycoproteins and glycopeptides from serum (65). Other studies compared lectin affinity and hydrazide chemistry methods for their efficiency in isolating glycoproteins and glycopeptides from a complex biological sample (39, 66, 67). The methods are complementary in enriching glycoproteins because of their different mechanisms of glycoprotein capturing. When both methods were applied, it significantly improves the coverage of the glycoproteome, resulting in an increased number of glycoproteins identified. The lectin affinity method can be tailored to target glycoproteins with specific glycan structure(s) for isolation using different lectins, thus, affording flexibility for its application in glycoproteomic studies. The application of hydrazide chemistry method has been widely used for N-linked glycosylation study. The hydrazide chemistry essentially reacts with all the proteins with carbonyl groups, which may include glycoproteins with oxidized glycans (37, 40) and other oxidized proteins that carry carbonyl groups (68–70). The high specificity of this method may mainly result from the specificity of PNGase F, the enzyme cleaving N-glycosidic bonds to release N-glycoproteins and peptides from the solid phase. This method affords high efficiency and specificity in enriching N-linked glycoproteins or glycopeptides from a complex sample, and can be easily incorporated into a proteomics workflow for integrated analysis. In addition to the lectin and hydrazide chemistry-based methods, it has been suggested that boronic acid-based solid phase extraction may also be useful for an overall glycoproteome enrichment (44, 45), on the basis of the evidence that boronic acid can form diester bonds with most glycans, including both N-linked and O-linked glycosylation (71).Mass Spectrometric Analysis of Glycoproteome
Mass spectrometry, because of its high sensitivity and selectivity, has been one of the most versatile and powerful tools in glycoprotein analysis, to identify the glycoproteins, evaluate glycosylation sites, and elucidate the oligosaccharide structures (56, 72, 73). The utility of a top-down approach (intact protein based proteomics analysis) (74) for glycoprotein characterization in a complex sample is still technically challenging with the current technology. The most versatile and widely used current glycoproteomics methods are based on characterizing glycopeptides generated by the digestion of glycoproteins, analyzing either deglycosylated glycopeptides or intact glycopeptides with glycan attachment, as illustrated in Fig. 1.The direct analysis of intact glycopeptides with carbohydrate attachments is complicated by the mixed information obtained from the fragment ion spectra, which may include fragment ions from the peptide backbone, the carbohydrate group and the combinations of both. Although it is technically challenging to comprehensively analyze intact glycopeptides in a global scale for a complex biological sample, complementary information regarding peptide backbone and glycan structure can likely be obtained in a single measurement. Early work using collision-induced dissociation (CID)1 has identified a few key features that are characteristics of the fragmentation of glycopeptides, providing the basis for intact glycopeptide identification (75–79). The analysis of intact glycopeptides has been carried out using a variety of different instruments, including electrospray ionization (EST)-based ion trap (IT) (80–84), quadrupole ion trap (QIT) (85–87), Fourier transform ion cyclotron resonance (FTICR) (31, 57, 88, 89), ion trap/time-of-flight (IT/TOF) (90, 91), and quadrupole/time-of-flight (Q/TOF) (92–97); matrix-assisted laser desorption/ionization (MALDI) based Q/TOF (98–100), quadrupole ion trap/time-of-flight (QIT/TOF) (86, 101, 102), and tandem time-of-flight (TOF/TOF) (81, 82, 101, 103–105) mass spectrometers. In general, the CID generated MS/MS spectrum of a glycopeptide is dominated by B- and Y-type glycosidic cleavage ions (carbohydrate fragments) (106), and b- and y-type peptide fragments from the peptide backbone. However, the MS/MS fragmentation data obtained from different instruments can have pronounced difference in providing structure information on glycan and peptide backbone, depending on the experimental setting and instrumentation used for mass analysis, including ionization methods, collision techniques and mass analyzers. Low energy CID with electrospray ionization-based ion trap, Fourier transform-ion cyclotron resonance, and Q/TOF instrument predominantly generates fragments of glycosidic bonds. The increase of collision energy using Fourier transform-ion cyclotron resonance, and Q/TOF instruments result in the more efficient fragmentation of b- and y- ions from the peptide backbone. MALDI ionization generates predominantly singly charged precursor ions, which are more stable and usually fragmented using higher energies via CID or post-source decay (PSD), generating fragments from both the peptide backbone and the glycan (98–100, 103, 107–110). Although Q/TOF instruments have been widely used for intact glycopeptide characterization, one unique feature of the ion trap instrument is that it allows repeated ion isolation/CID fragmentation cycles, which can provide a wealth of complementary information to interpret the structure of a glycan moiety and peptide backbone (56, 86, 111). Recently, fragmentation techniques using different mechanisms from CID have been introduced and applied for glycopeptide analysis, including infrared multiphoton dissociation (IRMPD) (112–115), electon-capture dissociation (ECD) (112–120) and electron-transfer disassociation (ETD) (85, 121–123). The application of infrared multiphoton dissociation and electon-capture dissociation is largely performed with Fourier transform-ion cyclotron resonance instruments. Complementary to CID fragmentation, electon-capture dissociation and electron-transfer disassociation tend to cleave the peptide backbone with no loss of the glycan moiety, providing specific information on localizing the glycosidic modification. More details regarding mass spectrometric analysis of intact glycopeptides can be found in recent reviews (56, 124). Although great efforts have been made to apply a variety of mass spectrometry techniques to study both N-linked (32, 56, 86, 87, 112–114, 125–130) and O-linked (90, 116, 119, 120, 130–140) glycopeptides, the interpretation of the fragment spectrum of an intact glycopeptide still requires intensive manual assignment and evaluation. A recent study has demonstrated the feasibility to develop an automated workflow for analyzing intact glycopeptides in mixtures (141). In general, however, a high throughput, large scale profiling of intact glycopeptides in a complex sample still remains a challenge with current technology.The analysis of deglycosylated peptides requires the removal of glycan attachments from glycopeptides. Fortunately, for N-linked glycopeptides, the N-glycosidic bond can be specifically cleaved using the enzyme PNGase F, providing deglycosylated peptides, which can then be analyzed directly using shotgun proteomics. The PNGase F-catalyzed deglycosylation results in the conversion of asparagine to aspartic acid in the glycopeptide sequence, which introduces a mass difference of 0.9840 Da. Such distinct mass differences can be used to precisely map the N-linked glycosylation sites using high resolution mass spectrometers. Stable isotope labeling introduced by enzymatic cleavage of glycans in H218O has also been used to enhance the precise identification of N-glycosylation sites (33, 142, 143). The removal of O-linked glycans is less straightforward, most assays rely on chemical deglycosylation methods, such as trifluoromethansulfonic acid (144), hydrazinolysis (145), β-elimination (146), and periodate oxidation (35, 147). The application of these methods suffers from a variety of limitations, such as low specificity for O-linked glycosylation, degradation of the peptide backbone, and modifications of the amino acid residues—all of which can complicate or compromise O-linked glycoproteomics analysis in a complex sample. Most of the large scale glycoproteomics studies using the deglycosylation approach have been focused on N-glycoproteins, which are prevalent in blood and a rich source for biomarker discovery. O-glycosylation lacks a common core, consensus sequence, and universal enzyme that can specifically remove the glycans from the peptide backbone, thus, is more challenging to analyze for large scale profiling.Following deglycosylation, the glycopeptides can be treated and analyzed as stripped peptides using a shotgun proteomics pipeline. MS/MS fragment spectra with b-ions and y-ions generated from CID are searched against protein databases using search algorithms, such as SEQUEST (148), MASCOT (149), and X!tandem (150), and subsequently validated via statistical analysis (151–154), to provide peptide and protein identifications with known false discovery rate. The N-glycosylation sites can be precisely mapped using the consensus sequence of Asn-X-Ser/Thr, in which asparagine is converted to aspartic acid following enzyme cleavage introducing a mass difference of 0.9840 Dalton. A variety of mass spectrometers have been used to analyze glycoproteins, in particular N-linked glycoproteins, in complex biological and clinical samples using the deglycosylation approach. These studies include electrospray ionization-based ion trap (37–39, 41, 67, 155–157), Orbitrap (158), Q/TOF (33, 35, 142, 155), triple quadrupole (159), Fourier transform-ion cyclotron resonance (64, 160); and MALDI based TOF/TOF (41, 161) and Q/TOF (37). Recently, an attempt was made to apply ion mobility-mass spectrometry (IM-MS) to characterize deglycosylated glycopeptides and the corresponding carbohydrates simultaneously (162) in a single measurement. The approach of analyzing deglycosylated glycopeptides makes it possible to utilize available proteomics technology for large-scale glycoproteome profiling, especially N-linked glycoproteins, in a high-throughput fashion.Glycoproteomics Analysis in Blood and Other Bodily Fluids
An important target for blood-based diagnostic assays involves the detection and quantification of glycosylated proteins. Glycosylated proteins, especially N-linked glycoproteins, are ubiquitous among the proteins destined for extracellular environments (163), such as plasma or serum. A systematic and in-depth global profiling of the blood glycoproteome can provide fundamental knowledge for blood biomarker development, and is now possible with the development of glycoproteomics technologies. In the past few years, several large scale proteomics studies on profiling the glycoproteome of human plasma and serum have been reported (34, 35, 37, 38, 43, 61, 65, 164–166), adding significant numbers of glycoproteins into the blood glycoproteome database. In one study (38), immunoaffinity subtraction and hydrazide chemistry were applied to enrich N-glycoproteins from human plasma. The captured plasma glycoproteins were subjected to two-dimensional liquid chromatography separation followed by tandem mass spectrometric analysis. A total of 2053 different N-glycopeptides were identified, covering 303 nonredundant glycoproteins, including many glycoproteins with low abundance in blood (38). In a different study, hydrazide chemistry-based solid phase extraction method was applied to enhance the detection of tissue-derived proteins in human plasma (167). Other studies have applied lectin affinity-based approaches to characterize the serum and plasma glycoproteome (34, 43, 166). These studies provide detailed identification regarding the individual N-glycosylation sites using high-resolution mass spectrometry. The efforts made in global profiling of glycoproteins in plasma and serum have not only greatly enhanced our understanding of the blood glycoproteome, but also have facilitated the development of new technologies that can be used for glycoprotein biomarker discovery. A variety of experimental designs and strategies for blood glycoprotein profiling have been applied for clinical disease studies, including prostate cancer (168), hepatocellular carcinoma (164, 168–170), lung adenocarcinoma (61, 171), breast cancer (58, 165, 172), atopic dermatitis (169), ovarian cancer (173, 174), congenital disorders of glycosylation (175), and pancreatic cancer (156, 176). Most of these studies focused on the early stages of glycoprotein biomarker discovery and many of them exploited multilectin affinity techniques to isolate glycoproteins from serum or plasma.Glycoproteomics techniques have also been applied to study the glycoproteome of other bodily fluids. The complementary application of hydrazide chemistry-based solid phase extraction and lectin affinity method have led to the identification of 216 glycoproteins in human cerebrospinal fluid (CSF), including many low abundant ones (39). A hydrazide chemistry based study on human saliva has characterized 84 N-glycosylated peptides in 45 glycoproteins (177). The study on tear fluid identified 43 N-linked glycoproteins, including 19 proteins that have not been discovered in tear fluid previously (178). Other glycoproteomics studies on bodily fluids include N-glycoprotein profiling of lung adenocarcinoma pleural effusions (179), urine glycoprotein profiling (180), and urine glycoprotein signature identification for bladder cancer (181). In the urine glycoprotein profiling study, 150 annotated glycoproteins in addition to 43 predicted glycoproteins were identified (180). In our own study, 48 glycoproteins have so far been identified in pancreatic juice (unpublished data), adding complementary information to the pancreatic juice protein database (182–184).Glycoproteomics Analysis of Tissue and Cell Lysates
Protein glycosylation has been increasingly recognized as one of the prominent alterations involved in tumorigenesis, inflammation, and other disease states. The study of glycoproteins in cell and tissue carries great promise for defining biomarkers for diagnotic and therapeutic targets. The glycoproteomics studies in liver tissue (185, 186) and cell lines (187) have provided a fundamental understanding of the liver glycoproteome and identified protein candidates that are associated with highly metastatic liver cancer cells. In one of the studies, hydrazide chemistry and multiple enzyme digestion provided a complementary identification of 939 N-glycosylation sites covering 523 nonredundant glycoproteins in human liver tissue (185). Studies on ovarian cancer have focused on discovering putative glycoprotein biomarkers for improving diagnosis (173, 174) and therapeutic treatment (188). Glycoproteomics studies have also been carried out to study hepatocelluar carcinoma. Magnetic nanoparticle immobilized Concanavalin A was used to selectively enrich N-glycoproteins in a hepatocelluar carcinoma cell line leading to the identification of 184 glycosylation sites corresponding to 101 glycoproteins (189). In a different study, complementary methods of hydrophilic affinity and hydrazide chemistry were applied to investigate the secreted glycoproteins from a hepatocelluar carcinoma cell line, in which 300 different glycosylation sites within 194 glycoproteins were identified (190). While many of these studies focused on N-glycoproteins, mucin-type O-linked glycoproteins are the predominant forms of O-linked glycosylation and are difficult to analyze. A metabolic labeling method was developed to facilitate their identification in complex cell lysates using proteomic strategies (191).Cell surface and membrane proteins are particularly appealing for biomarker discovery, and many of them are glycosylated proteins. Both hydrazide chemistry- and lectin affinity-based approaches have been applied to specifically study cell surface and membrane N-glycoproteins that are associated with diseases, including colon carcinoma (192), breast cancer (158), and thyroid cancer (157). One study applied hydrazide chemistry to covalently label extracellular glycan moieties on live cells, providing highly specific and selective identification of cell surface N-glycoproteins (64). A complementary application of hydrazide chemistry and lectin affinity methods was demonstrated to profile cell membrane glycoproteins, significantly enhancing the glycoprotein identification (67).Quantitative Glycoprotein Profiling
One of the major goals of clinical proteomics is to effectively identify dysregulated proteins that are specifically associated with a biological state, such as a disease. In the past decade, different quantitative proteomics techniques have been introduced and applied to study a wide variety of disease settings. These techniques are based on different mechanisms to facilitate mass spectrometric-based quantitative analysis, including stable isotopic or isobaric labeling using chemical reactions (e.g. ICAT and iTRAQ) (193–195), metabolic incorporation (e.g. SILAC) (196) and enzymatic reactions (e.g. 18O labeling) (197, 198); as well as less quantitatively accurate label-free approaches (199, 200). The overview and comparison of these quantitative techniques can be found in several reports in the literature and are not discussed in this review. Most of these isotopic labeling techniques can be adapted and utilized for glycoproteomics analysis to quantitatively compare the glycoproteome of a diseased sample to a control, thus revealing the glycosylation occupancy of individual glycosylation sites that may be involved in a disease. In addition to the well-established labeling methods cited above, several more experimental labeling strategies have been described in the field of glycoproteomics. One study demonstrated the feasibility of using stable isotope labeled succinic anhydride for quantitative analysis of glycoproteins isolated from serum via hydrazide chemistry (37). In a different report, the heavy and light version of N-acetoxy-succinimide combining with lectin affinity selection was used to quantitatively profile serum glycopeptides in canine lymphoma and transitional cell carcinoma (201). Stable isotope labeled 2-nitrobenzenesulfenyl was also used for chemical labeling in a quantitative glycoprotein profiling study on the sera from patients with lung adenocarcinoma (202). O-Linked N-acetylglucosamine (O-GlcNAc) is an intracellular, reversible form of glycosylation that shares many features with phosphorylation (203). Studies have suggested that O-GlcNAc may play an important role in many biological processes (204). A quantitative study on O-GlcNAc glycosylation has been reported, in which a method termed quantitative isotopic and chemoenzymatic tagging (QUIC-Tag) was described using a biotin-avidin affinity strategy for O-GlcNAc glycopeptide enrichment and stable isotope-labeled formaldehyde for mass spectrometric quantification (205). Recently, the isobaric tag for relative and absolute quantitation (iTRAQ) technique, combined with different glycoprotein enrichment approaches, has been utilized in several quantitative glycoproteomics studies. In the study of hepatocellular carcinoma, N-linked glycoproteins were enriched from hepatocellular carcinoma patients and controls using multilectin column and then quantitatively compared using iTRAQ to reveal the differential proteins associated with hepatocellular carcinoma (206). In a different study, the approach of using narrow selectivity lectin affinity chromatography followed by iTRAQ labeling was demonstrated to selectively identify differential glycoproteins in plasma samples from breast cancer patients (165). Another study utilized hydrazide chemistry-based solid phase extraction and iTRAQ to investigate the tear fluid of patients with climatic droplet keratopathy in comparison of normal controls, identifying multiple N-glycosylation sites with differential occupancy associated with climatic droplet keratopathy (178).In addition to using chemical reactions to incorporate stable isotope tag for quantitative mass spectrometric analysis, 18O can be introduced into N-glycopeptides during enzymatic reactions, such as tryptic digestion (incorporation of two 18O into the peptide carboxyl-terminal) and PNGase F mediated hydrolysis (incorporation of one 18O into the asparagine of N-glycosylation sites (33)). Attempts have been made to apply this approach to identify differentially expressed N-glycosylation associated with ovarian cancer in serum (207). In a different approach, the SILAC technique allows incorporation of stable isotope-labeled amino acids into proteins during cell culturing process (196), and was applied to investigate the difference in cell surface N-glycoproteins among different cell types (64). A label-free approach has also been used for glycoproteomics profiling, including a method developed to profile intact glycopeptides in a complex sample (208) and a study that compares the plasma glycoproteome between psoriasis patients and healthy controls (209).Targeted Glycoproteomics Analysis
Mass spectrometry based targeted proteomics has recently emerged as a multiplexed quantitative technique that affords highly specific and candidate-based detection of targeted peptides and proteins in a complex biological sample (18, 210–214). The technique is based on the concept of stable isotope dilution utilizing stable isotope-labeled synthetic reference peptides, which precisely mimic their endogenous counterparts, to achieve targeted quantification (214). Such techniques can be applied to target specific glycoproteins or glycopeptides, to precisely quantify the status of candidate glycosylation sites and assess the glycosylation occupancy at the molecular level. However, it is technically impractical to use synthetic peptides to precisely mimic a large number of natural glycopeptides with intact a glycan moiety as internal standards because of the structure complexity and variation of the sugar chain. To overcome these technical obstacles, an alternative approach was proposed for targeted analysis of N-glycosylation occupancy, in which stable isotope-labeled peptides were synthesized to mimic the deglycosylated form of candidate glycopeptides as internal references (161). It is known that the deglycosylation step using PNGase F results in a conversion of asparagine to aspartic acid in the peptide sequence, introducing a mass difference of 0.9840 Da. This phenomenon was utilized to design a synthetic peptide to mimic the endogenous N-linked glycopeptide in its deglycosylation form with exact amino acid sequence of its endogenous counterpart and with 13C and 15N labeling on one of its amino acids (161). Therefore, each matched pair of reference and endogenous candidate glycopeptides should share the same chromatographic and mass spectrometric characteristics, and can only be distinguished by their mass difference and isotopic pattern because of isotopic labeling. This design conceptually ensures that the synthetic internal standard of a candidate glycopeptide will be detected simultaneously with its endogenous form under the same analytical conditions, thus, minimizing the systematic variation and providing reliable quantification (214). The strategy for targeted glycoproteomics analysis is schematically illustrated in Fig. 2.Open in a separate windowFig. 2.Targeted analysis of N-glycopeptides.The targeted glycoproteomics technique was first demonstrated to analyze N-glycopeptides that were extracted from human serum using an integrated pipeline combining a hydrazide chemistry-based solid phase extraction method and a data-driven liquid chromatography MALDI TOF/TOF mass spectrometric analysis to quantify 21 N-glycopeptides in human serum (161). A similar mass spectrometric platform was then applied in a different study to assess a subset of glycoprotein biomarker candidates in the sera from prostate cancer patients (215). The targeted glycoproteomics analysis has also been demonstrated using a triple Q/linear ion trap instrument with the selected reaction monitoring (also referred to as multiple reaction monitoring) technique for highly sensitive targeted detection of N-glycoproteins in plasma (159). The technique was applied to detect tissue inhibitor of metalloproteinase 1 (TIMP1), an aberrant glycoprotein associated with colorectal cancer, in the sera of colorectal cancer patients (216) using a tandem enrichment strategy, combing lectin glycoprotein enrichment followed by the method of stable isotope standards and capture by antipeptide antibodies (SISCAPA), to enhance the detection of tissue inhibitor of metalloproteinase 1 (216). These studies demonstrate an integrated pipeline for candidate-based glycoproteomics analysis with precise mapping of targeted N-linked motifs and absolute quantification of the glycoprotein targets in a complex biological sample. Such targeted glycoproteomics can reach a detection sensitivity at the nanogram per milliliter level for serum and plasma detection (159, 214–216).Concluding Remarks
The major challenge for a comprehensive glycoproteomics analysis arises not only from the enormous complexity and nonlinear dynamic range in protein constituent in a clinical sample, but also the profound biological intricacy within the molecule of a glycoprotein, involving the flexibility in glycan structures and the complex linkage with the corresponding protein. In the past decade, significant efforts have been made to structurally or quantitatively characterize the glycoproteome of a variety of biological samples, and to investigate the significant glycoproteins in a wide assortment of diseases. Shotgun proteomics-based techniques are still the most effective and versatile approach in glycoproteomics analysis, allowing high throughput and detailed analysis on individual glycosylation sites. Although glycoproteomics is quickly emerging as an important technique for clinical proteomics study and biomarker discovery, a comprehensive, quantitative glycoproteomics analysis in a complex biological sample still remains challenging. It is anticipated that with the continued evolution in mass spectrometry, separation technology, and bioinformatics many of the technical limitations associated with current glycoproteomics may be transient. There is no doubt that glycoproteomics is playing an increasingly important role in biomarker discovery and clinical study. 相似文献5.
D. Friedecký P. Bednář M. Procházka T. Adam 《Nucleosides, nucleotides & nucleic acids》2013,32(9-11):1233-1236
A pilot study using capillary electrophoresis with mass spectrometry for the analysis of nucleotides in human erythrocytes is presented. Erythrocytes were incubated with 5-amino-4-imidazolecarboxamide riboside in order to mimic situation in defect of purine metabolism—AICA-ribosiduria. Characteristic AICA-ribotides together with normal nucleotides were separated by capillary electrophoresis in acetate buffer (20 mmol/L, pH 4.4) and identified on line by mass spectrometry. 相似文献
6.
Ngoc A. Dang Sjoukje Kuijper Elisabetta Walters Mareli Claassens Dick van Soolingen Gabriel Vivo-Truyols Hans-Gerd Janssen Arend H. J. Kolk 《PloS one》2013,8(10)
Tuberculosis (TB) remains a major international health problem. Rapid differentiation of Mycobacterium tuberculosis complex (MTB) from non-tuberculous mycobacteria (NTM) is critical for decisions regarding patient management and choice of therapeutic regimen. Recently we developed a 20-compound model to distinguish between MTB and NTM. It is based on thermally assisted hydrolysis and methylation gas chromatography-mass spectrometry and partial least square discriminant analysis. Here we report the validation of this model with two independent sample sets, one consisting of 39 MTB and 17 NTM isolates from the Netherlands, the other comprising 103 isolates (91 MTB and 12 NTM) from Stellenbosch, Cape Town, South Africa. All the MTB strains in the 56 Dutch samples were correctly identified and the model had a sensitivity of 100% and a specificity of 94%. For the South African samples the model had a sensitivity of 88% and specificity of 100%. Based on our model, we have developed a new decision-tree that allows the differentiation of MTB from NTM with 100% accuracy. Encouraged by these findings we will proceed with the development of a simple, rapid, affordable, high-throughput test to identify MTB directly in sputum. 相似文献
7.
Bettina Sarg Klaus Faserl Leopold Kremser Bernhard Halfinger Roberto Sebastiano Herbert H. Lindner 《Molecular & cellular proteomics : MCP》2013,12(9):2640-2656
We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano–liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and Nα-terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example.Histones are the most intensively studied group of basic nuclear proteins and are of great importance with regard to the organization of chromatin structure and control of gene activity. They are highly conserved during evolution, binding to and condensing eukaryotic chromosomal DNA to form chromatin. The fundamental chromatin subunit is the nucleosome, in which 166 bp of DNA are wrapped around a core histone octamer and a further ∼40 bp constitute the linker between one nucleosome core and the next. The histone octamer contains two molecules of each of the core histones H2A, H2B, H3, and H4. A fifth type of histone, referred to as linker histone (H1, H5), binds to both the DNA on the outer surface of nucleosomes and the linker DNA.There are numerous microsequence variants of linker and core histones (except H4) differing only slightly in primary sequence. In rat testis, for example, six somatic H1 subtypes, designated as H1a, H1b, H1c, H1d, H1e, and H1.0, as well as germ cell specific subtypes (i.e. H1t, H1T2, and HILS1), have been identified (1–3). Under various biological conditions, all histone proteins, for both linker and core histones, are subjected to post-translational modifications, including phosphorylation, acetylation, methylation, ubiquitination, deamidation, glycosylation, and ADP-ribosylation, which have a great influence on the epigenetic control of gene expression (4–6). The multitude of histone proteins resulting from closely related sequence variants and post-translational modifications, as well as their highly basic nature combined with hydrophobic properties, provides a major analytical challenge in current proteomics research. Over the past several years, considerable efforts have been expended to develop methods to identify the specific sites of histone modifications. Mass spectrometry (MS) coupled to liquid chromatography (LC) is the dominant technique for their characterization (7–14). However, because histone proteins contain up to nearly 35% basic amino acids, the analysis of histone peptides is still problematic, as digestion with many commonly used enzymes (e.g. trypsin, Lys-C, etc.) causes the formation of many short and polar peptides that poorly interact with the reverse-phase (RP)1 material and go undetected by conventional liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). To overcome this problem, chemical derivatization such as propionylation is often applied (15, 16).Capillary electrophoresis (CE) overcomes this disadvantage; this technique allows separations based on the mass-to-charge ratio of peptides and does not utilize their hydrophobic nature as a separation principle. The methods of electrophoresis and LC and their applicability for histone analysis have been reviewed in detail by Lindner (17). CE has proven to be a remarkably powerful method for separating individual histones and their modified forms based on their different electrophoretic mobilities. Using a bare fused silica capillary and hydroxypropylmethyl cellulose (HPMC) as a buffer additive in order to avoid undesired protein adsorption, different core and linker histones and their multiply phosphorylated and acetylated forms were successfully separated via capillary zone electrophoresis (CZE) (18–22). So far, no data have been published about the identification of histone modifications by means of capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS). LC is given preference over CE because of the difficulty of achieving on-line interfacing of CE with MS that allows stable electrospray processes without compromising the quality of separation or the detection sensitivity. However, CE-MS is a promising technique with constantly increasing importance, as documented by numerous articles (23–26).Various interfaces have been constructed to improve CESI-MS coupling (27, 28). Sheathflow interfaces are the most widely used, and although the drawback of having to dilute the analyte is inherent in this kind of interface, they offer stable electrophoretic separations and allow greater versatility in the choice of background electrolyte (BGE) and the range of flow rates (29–32). Sheathless interfaces have generated interest because no sheath liquid is added, which leads to enhanced detection sensitivity (33, 34). However, they have not been used frequently because of their limited robustness and lack of well-established interfaces and routine analysis protocols. The most widely used method for establishing the terminating electrical contact is coating the outer surface of the CE capillary tip with a conductive material (35–37). Unfortunately, the lifetimes of such coatings are generally very limited, as they suffer from deterioration under the influence of the high voltages applied.A recently published concept of a sheathless interface based on a separation capillary with a porous tip acting as a nanospray emitter overcomes these disadvantages (38). The capillary tip is etched using hydrofluoric acid until the capillary wall becomes so thin and porous that an electric contact can be established. The performance of this methodology, which combines the low-flow characteristics of CE with an integrated ESI source, is described in Refs. 39–41. Applications such as the analysis of intact proteins (42), protein–protein and protein–metal complexes (43), and ribosomal protein digests from E. coli (44) have been published. Method-inherent advantages of CESI-MS are highly efficient separations, low flow rates leading to reduced ion suppression, and greater sensitivity (40). In contrast to nano-LC, no column equilibration is needed, there are no gradient effects, and the instrumentation is less maintenance-intensive.Our group recently described important features of CESI-MS and reported the comparison of this method with LC-ESI-MS for the analysis of a 5% perchloric acid extraction of rat testis consisting mainly of different histone H1 subtypes (39). The performance of both techniques was evaluated regarding analysis time, protein sequence coverage, and number and molecular mass distribution of the identified peptides. The CESI-MS method provided shorter analysis times, narrower peaks yielding high signals, and the identification of a greater number of low molecular mass range peptides than LC-ESI-MS (39).In the current study, we investigated the analysis of post-translationally modified peptides, particularly phosphopeptides, obtained from endoproteinase Arg-C digested histones from rat testis; this organ contains the whole set of somatic and germ cell specific H1 histones, as well as numerous modified core histone proteins. CESI-MS and LC-ESI-MS were compared regarding the number and type of identified modified peptides. Without any pre-separation of the perchloric acid extraction, we found numerous known and novel modification sites in linker histones. In addition, immobilized metal-affinity chromatography (IMAC) experiments were utilized to enrich phosphopeptides prior to MS analysis. CESI-MS was also used for the rapid identification of post-translational modifications (PTMs) of rat testis core histones, which were pre-fractionated via RP-HPLC and digested with Arg-C. Using core histones from butyrate-treated mouse erythroleukemia cells, we further demonstrated that our method achieves excellent separations of intact histone subtypes and their multiply modified forms and enables the detection of the extent of PTMs in a fast and reproducible way. Our work represents the first detailed characterization of modified linker and core histone peptides and clearly demonstrates that CESI-MS is a promising alternative tool for epigenetic studies. 相似文献
8.
9.
Ma?a Sinreih Sven Zukunft Izidor Sosi? Jo?ko Cesar Stanislav Gobec Jerzy Adamski Tea Lani?nik Ri?ner 《PloS one》2015,10(2)
Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography—tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5α-pregnane-3-one and (20S)-5α-pregnane-3α,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing. 相似文献
10.
Ronda F Greaves 《The Clinical biochemist. Reviews / Australian Association of Clinical Biochemists》2012,33(4):123-132
Globally, harmonisation in laboratory medicine is a significant project. The relatively new implementation of liquid chromatography coupled with tandem mass spectrometry (LC-MSMS) techniques as routine assays in diagnostic laboratories provides the unique opportunity to harmonise, and in many cases standardise, methods from an early stage. This guide aims to provide a practical overview of the steps required to achieve agreement between LC-MSMS analytical procedures for routine clinical biochemistry diagnostic assays, with particular focus on the harmonisation and standardisation of methods currently implemented.To achieve harmonisation, and where practical standardisation, the approach is more efficient if divided into sequential stages. The suggested division entails: (i) planning and preliminary work; (ii) initial assessment of performance; (iii) standardisation and harmonisation initiative; (iv) establishing common reference intervals and critical limits; (v) developing best practice guidelines; and (vi) performing an ongoing review.The profession has a unique and significant opportunity to bring clinical mass spectrometry-based assays into agreement. Harmonisation of assays should ultimately provide the same result and interpretation for a given patient’s sample, irrespective of the laboratory that produced the result. To achieve this goal, we need to agree on the best practice LC-MSMS methods for use in routine clinical measurement. 相似文献
11.
Masao Ohnishi Masuo Nakano Yasuhiko Fujino 《Bioscience, biotechnology, and biochemistry》2013,77(3):743-744
Oxalate oxidase (EC 1.2.3.4) was purified to apparent homogeneity from Pseudomonas sp. OX-53. The molecular weight of the enzyme was about 320,000 by Sephadex G-200 column chromatography and 38,000 by sodium dodecyl sulfate disc electrophoresis. The isoelectric point of the enzyme was pH 4.7 by isoelectric focusing. This enzyme contained 1.12 atoms of manganese and 0.36 atoms of zinc per subunit. Besides oxalic acid, the enzyme oxidized glyoxylic acid and malic acid at lower reaction rates. The Michaelis constant of the enzyme was 9.5 mM for oxalic acid at the optimal pH 4.8. The enzyme was stable from pH 5.5 to 7.0. The enzyme was activated by flavins, phenylhydrazine, and o-phenylenediamine, and inhibited by I–, Br–, semicarbazide, and hydroxylamine. 相似文献
12.
13.
Dale?A. Shepherd Kris Holmes David?J. Rowlands Nicola?J. Stonehouse Alison?E. Ashcroft 《Biophysical journal》2013,105(5):1258-1267
The structural and functional analysis of the core protein of hepatitis B virus is important for a full understanding of the viral life cycle and the development of novel therapeutic agents. The majority of the core protein (CP149) comprises the capsid assembly domain, and the C-terminal region (residues 150–183) is responsible for nucleic acid binding. Protein monomers associate to form dimeric structural subunits, and helices 3 and 4 (residues 50–111 of the assembly domain) have been shown to be important for this as they constitute the interdimer interface. Here, using mass spectrometry coupled with ion mobility spectrometry, we demonstrate the conformational flexibility of the CP149 dimer. Limited proteolysis was used to locate involvement in this feature to the C-terminal region. A genetically fused CP dimer was found to show decreased disorder, consistent with a more restricted C-terminus at the fusion junction. Incubation of CP149 dimer with heteroaryldihydropyrimidine-1, a small molecule known to interfere with the assembly process, was shown to result in oligomers different in shape to the capsid assembly-competent oligomers of the fused CP dimer. We suggest that heteroaryldihydropyrimidine-1 affects the dynamics of CP149 dimer in solution, likely affecting the ratio between assembly active and inactive states. Therefore, assembly of the less dynamic fused dimer is less readily misdirected by heteroaryldihydropyrimidine-1. These studies of the flexibility and oligomerization properties of hepatitis B virus core protein illustrate both the importance of C-terminal dynamics in function and the utility of gas-phase techniques for structural and dynamical biomolecular analysis. 相似文献
14.
Dale A. Shepherd Kris HolmesDavid J. Rowlands Nicola J. Stonehouse Alison E. Ashcroft 《Biophysical journal》2013
The structural and functional analysis of the core protein of hepatitis B virus is important for a full understanding of the viral life cycle and the development of novel therapeutic agents. The majority of the core protein (CP149) comprises the capsid assembly domain, and the C-terminal region (residues 150–183) is responsible for nucleic acid binding. Protein monomers associate to form dimeric structural subunits, and helices 3 and 4 (residues 50–111 of the assembly domain) have been shown to be important for this as they constitute the interdimer interface. Here, using mass spectrometry coupled with ion mobility spectrometry, we demonstrate the conformational flexibility of the CP149 dimer. Limited proteolysis was used to locate involvement in this feature to the C-terminal region. A genetically fused CP dimer was found to show decreased disorder, consistent with a more restricted C-terminus at the fusion junction. Incubation of CP149 dimer with heteroaryldihydropyrimidine-1, a small molecule known to interfere with the assembly process, was shown to result in oligomers different in shape to the capsid assembly-competent oligomers of the fused CP dimer. We suggest that heteroaryldihydropyrimidine-1 affects the dynamics of CP149 dimer in solution, likely affecting the ratio between assembly active and inactive states. Therefore, assembly of the less dynamic fused dimer is less readily misdirected by heteroaryldihydropyrimidine-1. These studies of the flexibility and oligomerization properties of hepatitis B virus core protein illustrate both the importance of C-terminal dynamics in function and the utility of gas-phase techniques for structural and dynamical biomolecular analysis. 相似文献
15.
16.
17.
《Bioscience, biotechnology, and biochemistry》2013,77(8):2164-2169
Mass spectral measurements by electrospray ionization mass spectrometry (ESI-MS) detected the ions of β-cyclodextrin (βCD) or branched βCDs (glucosyl-, galactosyl-, mannosyl- and maltosyl-βCD)–prostaglandins (PGs: PGA2, PGD2, PGE1, PGE2, PGF2α and PGJ2) complexes, i.e., βCD–PG complexes, with a host:guest ratio of 1:1 in the negative ion mode. This is the first study to report the ions of branched βCD–PG complexes using ESI-MS. The inclusion complexes were determined by a flow injection analysis using acetonitrile/water. We could confirm by this method the presence of a βCD–PGE2 complex with a host:guest ratio of 1:1 in a solution-dissolved pharmaceutical formulation consisting of βCD–PGE2 (ProstarmonTM E tablet). 相似文献
18.
Mathias Walzer Da Qi Gerhard Mayer Julian Uszkoreit Martin Eisenacher Timo Sachsenberg Faviel F. Gonzalez-Galarza Jun Fan Conrad Bessant Eric W. Deutsch Florian Reisinger Juan Antonio Vizcaíno J. Alberto Medina-Aunon Juan Pablo Albar Oliver Kohlbacher Andrew R. Jones 《Molecular & cellular proteomics : MCP》2013,12(8):2332-2340
The range of heterogeneous approaches available for quantifying protein abundance via mass spectrometry (MS)1 leads to considerable challenges in modeling, archiving, exchanging, or submitting experimental data sets as supplemental material to journals. To date, there has been no widely accepted format for capturing the evidence trail of how quantitative analysis has been performed by software, for transferring data between software packages, or for submitting to public databases. In the context of the Proteomics Standards Initiative, we have developed the mzQuantML data standard. The standard can represent quantitative data about regions in two-dimensional retention time versus mass/charge space (called features), peptides, and proteins and protein groups (where there is ambiguity regarding peptide-to-protein inference), and it offers limited support for small molecule (metabolomic) data. The format has structures for representing replicate MS runs, grouping of replicates (for example, as study variables), and capturing the parameters used by software packages to arrive at these values. The format has the capability to reference other standards such as mzML and mzIdentML, and thus the evidence trail for the MS workflow as a whole can now be described. Several software implementations are available, and we encourage other bioinformatics groups to use mzQuantML as an input, internal, or output format for quantitative software and for structuring local repositories. All project resources are available in the public domain from the HUPO Proteomics Standards Initiative http://www.psidev.info/mzquantml.The Proteomics Standards Initiative (PSI) has been working for ten years to improve the reporting and standardization of proteomics data. The PSI has published minimum reporting guidelines, called MIAPE (Minimum Information about a Proteomics Experiment) documents, for MS-based proteomics (1) and molecular interactions (2), as well as data standards for raw/processed MS data in mzML (3), peptide and protein identifications in mzIdentML (4), transitions for selected reaction monitoring analysis in TraML (5), and molecular interactions in PSI-MI format (6). Standards are particularly important for quantitative proteomics research, because the associated bioinformatics analysis is highly challenging as a result of the range of different experimental techniques for deriving abundance values for proteins using MS. The techniques can be broadly divided into those based on (i) differential labeling, in which a metabolic label or chemical tag is applied to cells, peptides, or proteins, samples are mixed, and intensity signals for peptide ions are compared within single MS runs; or (ii) label-free methods in which MS runs occur in parallel and bioinformatics methods are used to extract intensity signals, ensuring that like-for-like signals are compared between runs (7). In most label-based and label-free approaches, peptide ratios or abundance values must be summarized in order for one to arrive at relative protein abundance values, taking into account ambiguity in peptide-to-protein inference. Absolute protein abundance values can typically be derived only using internal standards spiked into samples of known abundance (8, 9). The PSI has recently developed a MIAPE-Quant document defining and describing the minimal information necessary in order to judge or repeat a quantitative proteomics experiment.Software packages tend to report peptide or protein abundance values in a bespoke format, often as tab or comma separated values, for import into spreadsheet software. In complementary work, the PSI has developed a standard format for capturing these final results in a standardized tab separated value format, called mzTab, suitable for post-processing and visualization in end-user tools such as Microsoft Excel or the R programming language. The final results of a quantitative analysis are sufficient for many purposes, such as performing statistical analysis to determine differential expression or cluster analysis to find co-expressed proteins. However, mzTab (or similar bespoke formats) was not designed to hold a trace of how the peptide and protein abundance values were calculated from MS data (i.e. metadata is lost that might be crucial for other tasks). For example, most quantitative software packages detect and quantify so-called “features” (representing all ions collected for a given peptide) in two-dimensional MS data, where the two dimensions are retention time from liquid chromatography (LC) and mass over charge (m/z). Without capturing the two-dimensional coordinates of the features, it is not possible to write visualization software showing exactly what the software has quantified; researchers have to trust that the software has accurately quantified all ions from isotopes of a given peptide, excluding any overlapping ions derived from other peptides. The history of proteomics research has been one in which studies of highly variable quality have been published. There is also little quality control or benchmarking performed on quantitative software (10), meaning it is difficult to make quality judgments on a set of peptide and protein abundance values. The PSI has recently developed mzML, which can capture raw or processed MS data in a vendor neutral format, and the mzIdentML standard, to capture search engine results and the important metadata (such as software parameters), such that peptide and protein identification data can be interpreted consistently. These two standards are now being used for data sharing and to support open source software development, so that informatics groups can focus on algorithmic development rather than file format conversions. Until now, there has been no widely used open source format or data standard for capturing metadata and data relating to the quantitation step of analysis pipelines. In this work, we report the mzQuantML standard from the PSI, which has recently completed the PSI standardization process (11), from which version 1.0 was released. We believe that quantitative proteomics research will benefit from improved capabilities for tracing what manipulations have happened to data at each stage of the analysis process. The mzQuantML standard has been designed to store quantitative values calculated for features, peptides, proteins, and/or protein groups (where there is ambiguity in protein inference), plus associated software parameters. It has also been designed to accommodate small molecule data to improve interoperability with metabolomics investigations. The format can represent experimental replicates and grouping of replicates, and it has been designed via an open and transparent process. 相似文献
19.
David B. Nix Tadahiro Kumagai Toshihiko Katoh Michael Tiemeyer Kazuhiro Aoki 《Journal of visualized experiments : JoVE》2014,(93)
Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive β-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE. 相似文献
20.
Je-Hyun Baek Won Suk Yang Cheolju Lee Myeong-Hee Yu 《Molecular & cellular proteomics : MCP》2009,8(5):1072-1081
The native state of α1-antitrypsin (α1AT), a member of the serine protease inhibitor (serpin) family, is considered a kinetically trapped folding intermediate that converts to a more stable form upon complex formation with a target protease. Although previous structural and mutational studies of α1AT revealed the structural basis of the native strain and the kinetic trap, the mechanism of how the native molecule overcomes the kinetic barrier to reach the final stable conformation during complex formation remains unknown. We hypothesized that during complex formation, a substantial portion of the molecule undergoes unfolding, which we dubbed functional unfolding. Hydrogen-deuterium exchange coupled with ESI-MS was used to analyze this serpin in three forms: native, complexing, and complexed with bovine β-trypsin. Comparing the deuterium content at the corresponding regions of these three samples, we probed the unfolding of α1AT during complex formation. A substantial portion of the α1AT molecule unfolded transiently during complex formation, including not only the regions expected from previous structural studies, such as the reactive site loop, helix F, and the following loop, but also regions not predicted previously, such as helix A, strand 6 of β-sheet B, and the N terminus. Such unfolding of the native interactions may elevate the free energy level of the kinetically trapped native serpin sufficiently to cross the transition state during complex formation. In the current study, we provide evidence that protein unfolding has to accompany functional execution of the protein molecule.The native strain of serine protease inhibitors (serpins)1 is considered to be crucial to their biological functions, such as plasma protease inhibition (1, 2) and hormone delivery (3). Functional execution of serpins is accompanied by the conversion of the strained native structure into a more stable conformation (4). Because some of the strained native serpin structures are spontaneously converted into a more relaxed stable latent form under physiological conditions (5–7), the native structure is not the thermodynamically most stable conformation but is a kinetically trapped conformation. Upon binding a target protease, the scissile bond of the reactive site loop (RSL) is cleaved while the protease is covalently attached to the N-terminal part of the RSL (8, 9). During the conversion of the strained structure into the stable complex conformation (Fig. 1), RSL is inserted into the central β-sheet (A sheet) between strands 3 and 5 (s3A and s5A) to form strand 4 (s4A), and the covalently attached protease is concomitantly translocated to the opposite pole (10). Serpin inhibition occurs via a suicide substrate mechanism (4, 11, 12) in which serpins, upon binding proteases, partition between cleaved serpins and stable serpin-enzyme complexes.Open in a separate windowFig. 1.Structures of native α1AT and α1AT-trypsin complex. Left, structure of native α1AT (Protein Data Bank code 1QLP) illustrated with secondary structural elements (1). Right, structure of α1AT-trypsin complex (Protein Data Bank code 1EZX). The nine α-helices are colored dark gray, and the 16 β-strands are colored light gray.As a member of the serpin family, α1-antitrypsin (α1AT), which serves to modulate the activity of human leukocyte elastase in the lung, has been most extensively studied with regard to both structure and inhibition mechanism. Our previous studies with stabilizing mutations of α1AT showed that the native strain is distributed throughout the molecule and that various unfavorable structural motifs, such as hydrophobic packing, cavity in the core, and surface hydrophobic patch, appear to maintain the native strain (13, 14). Indeed stabilizing mutations localized in the region of RSL insertion during complex formation affected the inhibitory function individually by retarding the loop insertion (15). Mutations in other regions did not affect the inhibitory function individually, but collectively these mutations affected the inhibitory function when the stabilization effect reached a certain threshold (16). Maintaining the kinetic trap appears to require sustaining RSL at the hydrophobic β-barrel composed of sheet B and sheet C (B/C β-barrel) because the conversion into the stable latent conformation occurs by destabilization of the B/C β-barrel (6) as well as by the extension of RSL length (17). Thus, upon binding a target protease, RSL cleavage appears to induce a conformational conversion, and the resultant strain throughout the molecule facilitates the opening of β-sheet A and the insertion of the RSL, which is critical for the inhibitory pathway as opposed to the substrate pathway (10).Although these structural and mutational studies revealed the structural basis for maintaining the kinetic trap and its relation to the inhibitory mechanism, several questions still remain. For example, what structural changes does the native serpin molecule undergo to overcome the kinetic barrier and reach the final stable conformation during the complex formation? In the present study, to probe the structural process of overcoming the kinetic barrier during complex formation with a target protease, amide hydrogen exchange (hydrogen-deuterium exchange (H/D-EX)) was explored during the conversion of the native α1AT to the stable complex. H/D-EX coupled with ESI-MS is a powerful analytical tool for observing protein dynamics, transient conformational changes, and protein-protein interactions (18–22). These experiments demonstrated that transient structural unfolding occurred in many regions in the α1AT molecule during formation of the complex with β-trypsin, and some of this unfolding was unpredicted from previous structural studies. 相似文献