pI-based fractionation of serum proteomes versus anion exchange after enhancement of low-abundance proteins by means of peptide libraries

The pre-treatment of biological extracts with the aim of detecting very low-abundance proteins generates complexity requiring a proper fractionation. Therefore the success of identifying all newly detectable species depends on the selected fractionation methods.In this context and starting from a human serum, where the dynamic concentration range was reduced by means of a preliminary treatment with a combinatorial hexapeptide ligand library, we fractionated the sample using a novel method based on the differences in isoelectric points of proteins by means of Solid-State Buffers (SSB) associated with cation exchangers. The number of fractions was limited to four and was compared to a classical anion exchange method generating the same number of fractions.What was observed is that when using SSB technology the protein redundancy between fractions was significantly reduced compared to ion exchange fractionation allowing thus a better detection of novel species. The analysis of trypsinized protein fractions by nanoLC-MS/MS confirmed that the SSB technology used is more discriminant than anion exchange chromatography fractionation.A sample fractionation by SSB after the reduction of dynamic concentration range can be accomplished without either adjustment of pH and ionic strength or protein concentration and cleanup. Both advantages over either classical chromatography or isoelectric fractionations allow approaching the discovery of markers of interest under easier conditions applicable in a variety of fields of investigation.     

The amplified peptidome: the new treasure chest of candidate biomarkers

Mass spectrometric analysis of the low-molecular weight (LMW) range of the serum/plasma proteome is revealing the existence of large numbers of previously unknown peptides and protein fragments predicted to be derived from low-abundance proteins. This raises the question of why such low abundance molecules would be retained at detectable levels in the circulation, instead of being rapidly cleared and excreted. Theoretical models of biomarker production and association with serum carrier proteins have been developed to elucidate the mechanisms governing biomarker half-life in the bloodstream. These models predict that the vast majority of LMW biomarkers exist in association with circulating high molecular mass carrier proteins. Moreover, the total serum/plasma concentration of the biomarker is largely determined by the clearance rate of the carrier protein, not the free-phase biomarker clearance itself. These predictions have been verified experimentally using molecular mass fractionation of human serum before mass spectrometry sequence analysis. These principles have profound implications for biomarker discovery and measurement.     

Quantification of Cardiovascular Biomarkers in Patient Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution

Verification of candidate biomarkers requires specific assays to selectively detect and quantify target proteins in accessible biofluids. The primary objective of verification is to screen potential biomarkers to ensure that only the highest quality candidates from the discovery phase are taken forward into preclinical validation. Because antibody reagents for a clinical grade immunoassay often exist for a small number of candidates, alternative methodologies are required to credential new and unproven candidates in a statistically viable number of serum or plasma samples. Using multiple reaction monitoring coupled with stable isotope dilution MS, we developed quantitative, multiplexed assays in plasma for six proteins of clinical relevance to cardiac injury. The process described does not require antibodies for immunoaffinity enrichment of either proteins or peptides. Limits of detection and quantitation for each signature peptide used as surrogates for the target proteins were determined by the method of standard addition using synthetic peptides and plasma from a healthy donor. Limits of quantitation ranged from 2 to 15 ng/ml for most of the target proteins. Quantitative measurements were obtained for one to two signature peptides derived from each target protein, including low abundance protein markers of cardiac injury in the nanogram/milliliter range such as the cardiac troponins. Intra- and interassay coefficients of variation were predominantly <10 and 25%, respectively. The configured multiplex assay was then used to measure levels of these proteins across three time points in six patients undergoing alcohol septal ablation for hypertrophic obstructive cardiomyopathy. These results are the first demonstration of a multiplexed, MS-based assay for detection and quantification of changes in concentration of proteins associated with cardiac injury in the low nanogram/milliliter range. Our results also demonstrate that these assays retain the necessary precision, reproducibility, and sensitivity to be applied to novel and uncharacterized candidate biomarkers for verification of proteins in blood.Discovery of disease-specific biomarkers with diagnostic and prognostic utility has become an important challenge in clinical proteomics. In general, unbiased discovery experiments often result in the confident identification of thousands of proteins, hundreds of which may vary significantly between case and control samples in small discovery studies. However, because of the stochastic sampling of proteomes in discovery “omics” experiments, a large fraction of the protein biomarkers “discovered” in these experiments are false positives arising from biological or technical variability. Clearly discovery omics experiments do not lead to biomarkers of immediate clinical utility but rather produce candidates that must be qualified and verified in larger sample sets than were used for discovery (1).Traditional, clinical validation of biomarkers has relied primarily on immunoassays because of their specificity and sensitivity for the target analyte and high throughput capability. However, antibody reagents for a clinical grade immunoassay often only exist for a short list of candidates. The development of a reliable sandwich immunoassay for one target protein is expensive, has a long development time, and is dependent upon the generation of high quality protein antibodies. For the large majority of new, unproven candidate biomarkers, an intermediate verification technology is required that has shorter assay development time lines, lower assay cost, and effective multiplexing of dozens of candidates in low sample volumes. Ideally the approach should be capable of analyzing hundreds of samples of serum or plasma with good precision. The desired outcome of verification is a small number of highly credentialed candidates suitable for traditional preclinical and clinical validation studies.Multiple reaction monitoring (MRM)¹ coupled with stable isotope dilution (SID) MS has recently been shown to be well suited for direct quantification of proteins in plasma (2–4) and has emerged as the core technology for candidate biomarker verification. MRM assays can be highly multiplexed such that a moderate number of candidate proteins (in the range of 10–50) can be simultaneously targeted and measured in the statistically viable number of patient samples required for verification (hundreds of serum samples). However, sensitivity for unambiguous detection and quantification of proteins by MS-based assays is often constrained by sample complexity, particularly when the measurements are being made in complex fluids such as plasma.Many biomarkers of current clinical importance, such as prostate-specific antigen and the cardiac troponins, reside in the low nanogram/milliliter range in plasma and, until recently, have been inaccessible by non-antibody approaches. Our laboratory has recently shown for the first time that a combination of abundant protein depletion with limited fractionation at the peptide level prior to SID-MRM-MS provides robust limits of quantitation (LOQs) in the 1–20 ng/ml range with coefficient of variation (CV) of 10–20% at the LOQ for proteins in plasma (3).Here we demonstrate that this work flow can be extended to configure assays for a number of known markers of cardiovascular disease and, more importantly, can be deployed to measure their concentrations in clinical samples. We modeled a verification study comprising six patients undergoing alcohol septal ablation treatment for hypertrophic obstructive cardiomyopathy, a human model of “planned” myocardial infarction (PMI), and obtained targeted, quantitative measurements for moderate to low concentrations of cardiac biomarkers in plasma. This work provides additional evidence that MS-based assays can be configured and applied to verification of new protein targets for which high quality antibody reagents are not available.     

Efficient prefractionation of low-abundance proteins in human plasma and construction of a two-dimensional map

Human plasma is the most clinically valuable specimen, containing not only a dynamic concentration range of protein components, but also several groups of high-abundance proteins that seriously interfere with the detection of low-abundance potential biomarker proteins. To establish a high-throughput method for efficient depletion of high-abundance proteins and subsequent fractionation, prior to molecular analysis of proteins, we explored how coupled immunoaffinity columns, commercially available as multiple affinity removal columns (MARC) and free flow electrophoresis (FFE), could apply to the HUPO plasma proteome project. Here we report identification of proteins and construction of a human plasma 2-DE map devoid of six major abundance proteins (albumin, transferrin, IgG, IgA, haptoglobin, and antitrypsin) using MARC. The proteins were identified by PMF, matching with various internal 2-DE maps, resulting in a total of 144 nonredundant proteins that were identified from 398 spots. Tissue plasminogen activator, usually present at 10-60 ng/mL plasma, was also identified, indicative of a potentially low-abundance biomarker. Comparison of representative 2-D gel images of three ethnic groups (Caucasian, Asian-American, African-American) plasma exhibited minor differences in certain proteins between races and sample pretreatment. To establish a throughput fractionation of plasma samples by FFE, either MARC flow-through fractions or untreated samples of Korean serum were subjected to FFE. After separation of samples on FFE, an aliquot of each fraction was analyzed by 1-D gel, in which MARC separation was a prerequisite for FFE work. Thus, a working scheme of MARC --> FFE --> 1-D PAGE --> 2-D-nanoLC-MS/MS may be considered as a widely applicable standard platform technology for fractionation of complex samples like plasma.     

New approaches for biomarker discovery: the search for liver fibrosis markers in hepatitis C patients

Despite many shortcomings, liver biopsy is regarded as the gold standard for assessing liver fibrosis. A less invasive and equally or more reliable approach would constitute a major advancement in the field. Proteomics can aid discovery of novel serological markers and these proteins can be measured in patient blood. A major challenge of discovering biomarkers in serum is the presence of highly abundant serum proteins, which restricts the levels of total protein loaded onto gels and limits the detection of low abundance features. To overcome this problem, we used two-dimensional gel electrophoresis (2-DE) over a narrow pH 3-5.6 range since this lies outside the range of highly abundant albumin, transferrin and immunoglobulins. In addition, we used in-solution isoelectric focusing followed by SDS-PAGE to find biomarkers in hepatitis C induced liver cirrhosis. Using the pH 3-5.6 range for 2-DE, we achieved improved representation of low abundance features and enhanced separation. We found in-solution isoelectric focusing to be beneficial for analyzing basic, high molecular weight proteins. Using this method, the beta chains of both complement C3 and C4 were found to decrease in serum from hepatitis C patients with cirrhosis, a change not observed previously by 2-DE. We present two proteomics approaches that can aid in the discovery of clinical biomarkers in various diseases and discuss how these approaches have helped to identify 23 novel biomarkers for hepatic fibrosis.     

Human blood plasma preparation for two-dimensional gel electrophoresis

Human plasma consists of mainly large proteins, which vary in terms of both composition and concentration with the physiological state of the individual. Alterations in protein concentrations reflect the current state of the individual's health and thus may be utilized as valuable biomarkers for a specific biological process or disease. Two-dimensional gel electrophoresis (2-DE) has proven to be a valuable method for the separation and comparison of complex protein mixtures, for example, from disease and healthy states, as this method provides information regarding the variation, relative quantities, and structures of the intact proteins. The procedures utilized for the preparation of samples for 2-DE are critical to the acquisition of high-quality results for the discovery of biomarkers. The objective of this study was to review the preparation methods of plasma for 2-DE, particularly those designed to improve the detection of proteins in low abundance in plasma on 2-DE. The use of anticoagulants and protease inhibitors during the collection of blood, the removal of abundant proteins using multicomponent immunodepletion system, and desalting procedure allow us to compile profiles of proteins occurring in low concentrations in the plasma and to improve the pattern generated during 2-DE.     

Comparison of alternative analytical techniques for the characterisation of the human serum proteome in HUPO Plasma Proteome Project

Based on the same HUPO reference specimen (C1-serum) with the six proteins of highest abundance depleted by immunoaffinity chromatography, we have compared five proteomics approaches, which were (1) intact protein fractionation by anion-exchange chromatography followed by 2-DE-MALDI-TOF-MS/MS for protein identification (2-DE strategy); (2) intact protein fractionation by 2-D HPLC followed by tryptic digestion of each fraction and microcapillary RP-HPLC/microESI-MS/MS identification (protein 2-D HPLC fractionation strategy); (3) protein digestion followed by automated online microcapillary 2-D HPLC (strong cation-exchange chromatography (SCX)-RPC) with IT microESI-MS/MS; (online shotgun strategy); (4) same as (3) with the SCX step performed offline (offline shotgun strategy) and (5) same as (4) with the SCX fractions reanalysed by optimised nanoRP-HPLC-nanoESI-MS/MS (offline shotgun-nanospray strategy). All five approaches yielded complementary sets of protein identifications. The total number of unique proteins identified by each of these five approaches was (1) 78, (2) 179, (3) 131, (4) 224 and (5) 330 respectively. In all, 560 unique proteins were identified. One hundred and sixty-five proteins were identified through two or more peptides, which could be considered a high-confidence identification. Only 37 proteins were identified by all five approaches. The 2-DE approach yielded more information on the pI-altered isoforms of some serum proteins and the relative abundance of identified proteins. The protein prefractionation strategy slightly improved the capacity to detect proteins of lower abundance. Optimising the separation at the peptide level and improving the detection sensitivity of ESI-MS/MS were more effective than fractionation of intact proteins in increasing the total number of proteins identified. Overall, electrophoresis and chromatography, coupled respectively with MALDI-TOF/TOF-MS and ESI-MS/MS, identified complementary sets of serum proteins.     

High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites

The detection and quantification of plasma (serum) proteins at or below the ng/ml concentration range are of critical importance for the discovery and evaluation of new protein biomarkers. This has been achieved either by the development of high sensitivity ELISA or other immunoassays for specific proteins or by the extensive fractionation of the plasma proteome followed by the mass spectrometric analysis of the resulting fractions. The first approach is limited by the high cost and time investment for assay development and the requirement of a validated target. The second, although reasonably comprehensive and unbiased, is limited by sample throughput. Here we describe a method for the detection of plasma proteins at concentrations in the ng/ml or sub-ng/ml range and their accurate quantification over 5 orders of magnitude. The method is based on the selective isolation of N-glycosites from the plasma proteome and the detection and quantification of targeted peptides in a quadrupole linear ion trap instrument operated in the multiple reaction monitoring (MRM) mode. The unprecedented sensitivity of the mass spectrometric analysis of minimally fractionated plasma samples is the result of the significantly reduced sample complexity of the isolated N-glycosites compared with whole plasma proteome digests and the selectivity of the MRM process. Precise quantification was achieved via stable isotope dilution by adding (13)C- and/or (15)N-labeled reference analytes. We also demonstrate the possibility of significantly expanding the number of MRM measurements during one single LC-MS run without compromising sensitivity by including elution time constraints for the targeted transitions, thus allowing quantification of large sets of peptides in a single analysis.     

High dynamic range characterization of the trauma patient plasma proteome

Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this "divide-and-conquer" strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3,654 different proteins with 1,494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 "classic" cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2,910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1,553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.     

Affinity chromatography: a useful tool in proteomics studies

Separation or fractionation of a biological sample in order to reduce its complexity is often a prerequisite to qualitative or quantitative proteomic approaches. Affinity chromatography is an efficient protein separation method based on the interaction between target proteins and specific immobilized ligands. The large range of available ligands allows to separate a complex biological extract in different protein classes or to isolate the low abundance species such as post-translationally modified proteins. This method plays an essential role in the isolation of protein complexes and in the identification of protein-protein interaction networks. Affinity chromatography is also required for quantification of protein expression by using isotope-coded affinity tags.     

Analysis of the human serum proteome

Changes in serum proteins that signal histopathological states, such as cancer, are useful diagnostic and prognostic biomarkers. Unfortunately, the large dynamic concentration range of proteins in serum makes it a challenging proteome to effectively characterize. Typically, methods to deplete highly abundant proteins to decrease this dynamic protein concentration range are employed, yet such depletion results in removal of important low abundant proteins. A multi-dimensional peptide separation strategy utilizing conventional separation techniques combined with tandem mass spectrometry (MS/MS) was employed for a proteome analysis of human serum. Serum proteins were digested with trypsin and resolved into 20 fractions by ampholyte-free liquid phase isoelectric focusing. These 20 peptide fractions were further fractionated by strong cation-exchange chromatography, each of which was analyzed by microcapillary reversed-phase liquid chromatography coupled online with MS/MS analysis. This investigation resulted in the identification of 1444 unique proteins in serum. Proteins from all functional classes, cellular localization, and abundance levels were identified. This study illustrates that a majority of lower abundance proteins identified in serum are present as secreted or shed species by cells as a result of signalling, necrosis, apoptosis, and hemolysis. These findings show that the protein content of serum is quite reflective of the overall profile of the human organism and a conventional multidimensional fractionation strategy combined with MS/MS is entirely capable of characterizing a significant fraction of the serum proteome. We have constructed a publicly available human serum proteomic database (http://bpp.nci.nih.gov) to provide a reference resource to facilitate future investigations of the vast archive of pathophysiological content in serum. These authors contributed equally to this work.     

Improvement of the two-dimensional gel electrophoresis analysis for the proteome study of Halobacterium salinarum

Inherent problems exist in the use of two-dimensional gel electrophoresis (2-DE) for sample preparation and separation of proteins from Halobacterium salinarum. In particular, proteins from cells grown in 25% NaCl are difficult to resolve by 2-DE due to the abundance of salt. To remove salts, a 3 kDa molecular weight cut-off column was used. When soluble proteins were separated by 2-DE, most of the proteins were concentrated in the acidic range. For separation of proteins in the pH 3-6 range, ultrazoom immobilized pH gradient strips were used. In addition, sample separation using a IPGphor/Multiphor combined system was a more effective method for the proteome analysis of acidic proteins than using IPGphor for the isoelectric focusing step.     

Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots

The abundance profile of the human urinary proteome is known to change as a result of diseases or drug toxicities, particularly of those affecting the kidney and the urogenital tract. A consequence of such insults is the ability to identify proteins in urine, which may be useful as quantitative biomarkers. To succeed in discovering them, reproducible urine sample preparation methods and good protein resolution in two-dimensional electrophoresis (2-DE) gels for parallel semiquantitative protein measurements are desirable. Here, we describe a protein fractionation strategy enriching proteins of molecular masses (M(r)) lower than 30 kDa in a fraction separate from larger proteins. The fraction containing proteins with M(r)s higher than 30 kDa was subsequently subjected to immunoaffinity subtraction chromatography removing most of the highly abundant albumin and immunoglobulin G. Following 2-DE display, superior protein spot resolution was observed. Subsequent high-throughput mass spectrometry analysis of ca. 1400 distinct spots using matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting and liquid chromatography-electrospray ionization tandem mass spectrometry lead to the successful identification of 30% of the proteins. As expected from high levels of post-translational modifications in most urinary proteins and the presence of proteolytic products, ca. 420 identified spots collapsed into 150 unique protein annotations. Only a third of the proteins identified in this study are described as classical plasma proteins in circulation, which are known to be relatively abundant in urine despite their retention to a large extent in the glomerular blood filtration process. As a proof of principle that our urinary proteome display effort holds promise for biomarker discovery, proteins isolated from the urine of a renal cell carcinoma patient were profiled prior to and after nephrectomy. Particularly, the decrease in abundance of the kininogen 2-DE gel spot train in urine after surgery was striking.     

Immunoaffinity separation of plasma proteins by IgY microbeads: meeting the needs of proteomic sample preparation and analysis

Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95-99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein-protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics.     

Strategies for revealing lower abundance proteins in two-dimensional protein maps

One of the most challenging contemporary research endeavors is the mapping of proteins and establishing their linkages to normal and pathological conditions. The availability of current proteomics technologies has greatly facilitated the separation and identification of proteins in a complex protein mixture by standard two-dimensional gel electrophoresis and subsequent MALDI-TOF mass spectrometry. Due to the huge differences in the distribution of proteins in complex proteomes of humans, the detection and identification of proteins expressed in low copy number is a major challenge. The low abundance of important physiologically relevant proteins has rendered their analyses almost impossible without some means of prior purification and enrichment from tissue lysates or biological fluids. It is the current limits of detection of the methods that are used that prevents the detection of these proteins not the proteins themselves. More importantly, considering the frequency at which post-translational modifications of proteins occur, the separation of protein isoforms is essential to understand biological changes, and two-dimensional gel electrophoresis remains the only technique that can offer sufficient resolution to address this issue at a functional level. Cellular fractionation techniques followed by specific affinity probes for tracking target proteins have been developed to deplete the proteome of high abundance proteins in order to increase the sample loading for achieving greater sensitivity for proteins present in low abundance. Those applications can entail the removal of one protein or a class of proteins that interferes with the resolution of proteins in a 2-DE map. Moreover, the use of better solubilizing detergents in combination with an overlapping narrow immobilized pH gradients, results in higher resolution by stretching the protein pattern in the first dimension. In this review we will discuss strategies to remove high abundance proteins that can result in the visualization and detection of low abundance proteins in biological samples. The potential use of these strategies, as a means of developing diagnostic tools for early screening of diseases and identification of drug targets for therapeutic intervention, will also be discussed.     

Multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome

In order to discover novel protein markers indicative of disease processes or drug effects, the proteomics technology platform most commonly used consists of high resolution protein separation by two-dimensional electrophoresis (2-DE), mass spectrometric identification of proteins from stained gel spots and a bioinformatic data analysis process supported by statistics. This approach has been more successful in profiling proteins and their disease- or treatment-related quantitative changes in tissue homogenates than in plasma samples. Plasma protein display and quantitation suffer from several disadvantages: very high abundance of a few proteins; high heterogeneity of many proteins resulting in long charge trains; crowding of 2-DE separated protein spots in the molecular mass range between 45-80 kD and in the isoelectric point range between 4.5 and 6. Therefore, proteomic technologies are needed that address these problems and particularly allow accurate quantitation of a larger number of less abundant proteins in plasma and other body fluids. The immunoaffinity-based protein subtraction chromatography (IASC) described here removes multiple proteins present in plasma and serum in high concentrations effectively and reproducibly. Applying IASC as an upfront plasma sample preparation process for 2-DE, the protein spot pattern observed in gels changes dramatically and at least 350 additional lower abundance proteins are visualized. Affinity-purified polyclonal antibodies (pAbs) are the immunoaffinity reagents used to specifically remove the abundant proteins such as albumin, immunoglobulin G, immunoglobulin A, transferrin, haptoglobin, alpha-1-antitrypsin, hemopexin, transthyretin, alpha-2-HS glycoprotein, alpha-1-acid glycoprotein, alpha-2-macroglobulin and fibrinogen from human plasma samples. To render the immunoaffinity subtraction procedure recyclable, the pAbs are immobilized and cross-linked on chromatographic matrices. Antibody-coupled matrices specific for one protein each can be pooled to form mixed-bed IASC columns. We show that up to ten affinity-bound plasma proteins with similar solubility characteristics are eluted from a mixed-bed column in one step. This facilitates automated chromatographic processing of plasma samples in high throughput, which is desirable in proteomic disease marker discovery projects.     

Optimization of Plasma Sample Pretreatment for Quantitative Analysis Using iTRAQ Labeling and LC-MALDI-TOF/TOF

Shotgun proteomic methods involving iTRAQ (isobaric tags for relative and absolute quantitation) peptide labeling facilitate quantitative analyses of proteomes and searches for useful biomarkers. However, the plasma proteome''s complexity and the highly dynamic plasma protein concentration range limit the ability of conventional approaches to analyze and identify a large number of proteins, including useful biomarkers. The goal of this paper is to elucidate the best approach for plasma sample pretreatment for MS- and iTRAQ-based analyses. Here, we systematically compared four approaches, which include centrifugal ultrafiltration, SCX chromatography with fractionation, affinity depletion, and plasma without fractionation, to reduce plasma sample complexity. We generated an optimized protocol for quantitative protein analysis using iTRAQ reagents and an UltrafleXtreme (Bruker Daltonics) MALDI TOF/TOF mass spectrometer. Moreover, we used a simple, rapid, efficient, but inexpensive sample pretreatment technique that generated an optimal opportunity for biomarker discovery. We discuss the results from the four sample pretreatment approaches and conclude that SCX chromatography without affinity depletion is the best plasma sample preparation pretreatment method for proteome analysis. Using this technique, we identified 1,780 unique proteins, including 1,427 that were quantified by iTRAQ with high reproducibility and accuracy.     

Clinical Quantitation of Prostate-specific Antigen Biomarker in the Low Nanogram/Milliliter Range by Conventional Bore Liquid Chromatography-Tandem Mass Spectrometry (Multiple Reaction Monitoring) Coupling and Correlation with ELISA Tests

Proteomics discovery leads to a list of potential protein biomarkers that have to be subsequently verified and validated with a statistically viable number of patients. Although the most sensitive, the development of an ELISA test is time-consuming when antibodies are not available and need to be conceived. Mass spectrometry analysis driven in quantitative multiple reaction monitoring mode is now appearing as a promising alternative to quantify proteins in biological fluids. However, all the studies published to date describe limits of quantitation in the low μg/ml range when no immunoenrichment of the target protein is applied, whereas the concentration of known clinical biomarkers is usually in the ng/ml range. Using prostate-specific antigen as a model biomarker, we now provide proof of principle that mass spectrometry enables protein quantitation in a concentration range of clinical interest without immunoenrichment. We have developed and optimized a robust sample processing method combining albumin depletion, trypsin digestion, and solid phase extraction of the proteotypic peptides starting from only 100 μl of serum. For analysis, mass spectrometry was coupled to a conventional liquid chromatography system using a 2-mm-internal diameter reverse phase column. This mass spectrometry-based strategy was applied to the quantitation of prostate-specific antigen in sera of patients with either benign prostate hyperplasia or prostate cancer. The quantitation was performed against an external calibration curve by interpolation, and results showed good correlation with existing ELISA tests applied to the same samples. This strategy might now be implemented in any clinical laboratory or certified company for further evaluation of any putative biomarker in the low ng/ml range of serum or plasma.Used for years across a wide range of pathologies, proteomics studies based on semiquantitative mass spectrometry of proteins have already led to the discovery of numerous protein biomarker candidates. Often tens of putative biomarkers have been described for a single disease, but the subsequent phase of clinical evaluation on large cohorts for each candidate is clearly the bottleneck as revealed by the meager number of newly approved biomarkers for clinical use. One of the critical limitations of discovery work flows arises when no antibody is available to initiate an immunoassay because 1–3 years are required to conceive de novo a reliable immunoassay. Such a delay is a serious drawback when tens of putative markers are concerned. Quantitation of small organic molecules by mass spectrometry has been used extensively for years in the field of environmental contaminant analysis or pharmacokinetic profiling of drug candidates during clinical studies. More recently, absolute quantitation of proteins using mass spectrometry by single (SRM)¹ or multiple reaction monitoring (MRM) and stable isotope dilution has thus naturally emerged as an alternative to immunoassays. Basically the absolute quantitation of a protein is provided by the integration of the specific MRM signals corresponding, respectively, to a proteotypic peptide (1) obtained from enzymatic hydrolysis of the target protein (usually by trypsin) and to its synthetic stable isotope-labeled isotopomer (2). The validation criteria of an MS-based method in terms of accuracy and precision are relatively easy to fulfill when addressing small molecules or proteins below 10 kDa in plasma or serum. Indeed they may be easily extracted from the bulk of high molecular mass proteins simply by selective precipitation. The quantitation of higher molecular mass proteins has proven to be more challenging because of the complexity and large dynamic range of proteins in e.g. plasma. In a pioneering study Anderson and Hunter (3) successfully demonstrated generation of a multiplexed assay for proteins covering high (tens to hundreds of micrograms/milliliter) to medium (hundreds of nanograms/milliliter to a few micrograms/milliliter) abundance ranges in plasma when combined with immunodepletion. However, these ranges remain problematic because clinically relevant biomarkers are usually present in plasma or serum in the low nanogram/milliliter range or below. To significantly improve the limit of quantitation (LOQ) of LC-MRM mass spectrometry, Keshishian et al. (4) evaluated a combination of immunodepletion of the most abundant plasma or serum proteins with strong cation exchange (SCX) chromatography for sample preparation prior to LC-MRM analysis. LOQs in the 1–10 ng/ml range were obtained with a coefficient of variation from 3 to 15% for five exogenous non-human proteins and the human prostate-specific antigen (PSA) protein spiked together into immunodepleted plasma from a healthy female donor. Very recently, a new approach using product ion scanning on a linear ion trap was proposed by Diamandis and co-workers (5) that allowed reaching a limit of quantitation of 1 ng/ml for PSA spiked into control plasma. This study marked a gain in sensitivity compared with previous attempts by others on similar instrumentation (6–9) but applied immunopurification of the target protein.Interestingly all the strategies published to date have been dealing with analytical development of work flows for the validation of biomarker candidates using microbore, nanoflow chromatography. Nanoflow is without any doubt appealing over conventional microflow during the proteomics discovery phase when the amount of biological material, for instance from a tumor biopsy, is often limited. Nonetheless this technique inherently still suffers from a lack of robustness and requires skilled personnel to be operational on a daily basis. As a consequence, nanoflow chromatography is not easily adaptable for the high throughput analysis environment encountered in clinical laboratories or good laboratory practice-certified or contract research organization companies where hundreds of samples are handled per day. In such organizations only microflow separations using 1- or 2-mm-internal diameter HPLC columns are compatible with the requirements of robustness and sample throughput.Therefore, the present work was centered on how a simple work flow could, in the near future, enable the large scale verification phase of putative biomarkers in the ng/ml of plasma range by the use of conventional LC equipment, i.e. using a 2-mm-internal diameter HPLC column. To address this question, we have considered that the absolute quantitation of PSA in true clinical samples could represent a challenging model. Combining immunodepletion of serum albumin and peptide fractionation simply by solid phase extraction (SPE), we were able to demonstrate for the first time the absolute quantitation of PSA by LC-MRM mass spectrometry in clinical serum samples of patients with benign prostate hyperplasia (BPH) or prostate cancer (PCa) within concentrations ranging from 4 to 30 ng/ml. Furthermore a good correlation was observed between the clinical ELISA tests and the mass spectrometry-based assays. We believe that these results are an unprecedented demonstration that the clinical relevance of putative biomarkers issued from proteomics investigation may now be confidently evaluated in the ng/ml range by robust coupling between conventional bore LC and mass spectrometry.     

Extraction of wheat endosperm proteins for proteome analysis

Total protein extracts of wheat endosperm are widely used for the analysis of the highly abundant gliadins and glutenins. In this review, the most popular total endosperm extraction methods are compared for their effectiveness in proteome coverage. A drawback of total endosperm extracts is that the enormous dynamic range of protein abundance limits the detection, quantification, and identification of low abundance proteins. Protein fractionation is invaluable for improving proteome coverage, because it reduces sample complexity while enriching for specific classes of less abundant proteins. A wide array of techniques is available for isolating protein subpopulations. Sequential extraction is a method particularly suited for subfractionation of wheat endosperm proteins, because it takes advantage of the specific solubility properties of the different classes of endosperm proteins. This method effectively separates the highly abundant gliadins and glutenins from the much less abundant albumins and globulins. Subcellular fractionation of tissue homogenates is a classical technique for isolating membranes and organelles for functional analysis. This approach is suitable for defining the biochemical processes associated with amyloplasts, specialized organelles in the endosperm that function in the synthesis and storage of starch. Subproteome fractionation, when combined with 2-DE and protein identification, provides a powerful approach for defining endosperm protein composition and providing new insights into cellular functions.     

A novel four-dimensional strategy combining protein and peptide separation methods enables detection of low-abundance proteins in human plasma and serum proteomes

A novel strategy, termed protein array pixelation, is described for comprehensive profiling of human plasma and serum proteomes. This strategy consists of three sequential high-resolution protein prefractionation methods (major protein depletion, solution isoelectrofocusing, and 1-DE) followed by nanocapillary RP tryptic peptide separation prior to MS/MS analysis. The analysis generates a 2-D protein array where each pixel in the array contains a group of proteins with known pI and molecular weight range. Analysis of the HUPO samples using this strategy resulted in 575 and 2890 protein identifications from plasma and serum, respectively, based on HUPO-approved criteria for high-confidence protein assignments. Most importantly, a substantial number of low-abundance proteins (low ng/mL - pg/mL range) were identified. Although larger volumes were used in initial prefractionation steps, the protein identifications were derived from fractions equivalent to approximately 0.6 microL (45 microg) of plasma and 2.4 microL (204 microg) of serum. The time required for analyzing the entire protein array for each sample is comparable to some published shotgun analyses of plasma and serum proteomes. Therefore, protein array pixelation is a highly sensitive method capable of detecting proteins differing in abundance by up to nine orders of magnitude. With further refinement, this method has the potential for even higher capacity and higher throughput.