Quantification of seminolipid by LC-ESI-MS/MS-multiple reaction monitoring: compensatory levels in Cgt mice

Seminolipid, also known as sulfogalactosylglycerolipid (SGG), plays important roles in male reproduction. Therefore, an accurate and sensitive method for SGG quantification in testes and sperm is needed. Here we compare SGG quantitation by the traditional colorimetric Azure A assay with LC-ESI-MS/MS using multiple reaction monitoring (MRM). Inclusion of deuterated SGG as the internal standard endowed accuracy to the MRM method. The results showed reasonable agreement between the two procedures for purified samples, but for crude lipid extracts, the colorimetric assay significantly overestimated the SGG content. Using ESI-MS/MS MRM, C16:0-alkyl/C16:0-acyl SGG of Cgt^+/− mice was quantified to be 406.06 ± 23.63 μg/g testis and 0.13 ± 0.02 μg/million sperm, corresponding to 78% and 87% of the wild-type values, respectively. CGT (ceramide galactosyltransferase) is a critical enzyme in the SGG biosynthesis pathway. Cgt^−/− males depleted of SGG are infertile due to spermatogenesis arrest. However, Cgt^+/− males sire offspring. The higher than 50% expression level of SGG in Cgt^+/− animals, compared with the wild-type expression, might be partly due to compensatory translation of the active CGT enzyme. The results also indicated that 78% of SGG levels in Cgt^+/− mice were sufficient for normal spermatogenesis.     

Targeted analysis of ganglioside and sulfatide molecular species by LC/ESI-MS/MS with theoretically expanded multiple reaction monitoring

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has been applied primarily to the analysis of glycosphingolipids separated from other complex mixtures by TLC, but it is difficult to obtain quantitative profiling of each glycosphingolipid among the different spots on TLC by MALDI-MS. Thus, the development of a convenient approach that utilizes liquid chromatography/electrospray ionization (LC/ESI)-MS has received interest. However, previously reported methods have been insufficient to separate and distinguish each ganglioside class. Here we report an effective method for the targeted analysis of theoretically expected ganglioside molecular species by LC/ESI tandem mass spectrometry (LC/ESI-MS/MS) in combination with multiple reaction monitoring (MRM). MRM detection specific for sialic acid enabled us to analyze ganglioside standards such as GM1, GM2, GM3, GD1, and GT1 at picomolar to femtomolar levels. Furthermore, other gangliosides, such as GD2, GD3, GT2, GT3, and GQ1, were also detected in glycosphingolipid standard mixtures from porcine brain and acidic glycolipid extract from mouse brain by theoretically expanded MRM. We found that this approach was also applicable to sulfatides contained in the glycosphingolipid mixtures. In addition, we established a method to separate and distinguish regioisomeric gangliosides, such as GM1a and -1b, GD1a, -1b, and -1c, and GT1a, -1b, and -1c with diagnostic sugar chains in the MRM.     

Improved analysis of bile acids in tissues and intestinal contents of rats using LC/ESI-MS

To evaluate bile acid (BA) metabolism in detail, we established a method for analyzing BA composition in various tissues and intestinal contents using ultra performance liquid chromatography/electrospray ionization mass spectrometry (UPLC/ESI-MS). Twenty-two individual BAs were determined simultaneously from extracts. We applied this method to define the differences in BA metabolism between two rat strains, WKAH and DA. The amount of total bile acids (TBAs) in the liver was significantly higher in WKAH than in DA rats. In contrast, TBA concentration in jejunal content, cecal content, colorectal content, and feces was higher in DA rats than in WKAH rats. Nearly all BAs in the liver were in the taurine- or glycine-conjugated form in DA rats, and the proportion of conjugated liver BAs was up to 75% in WKAH rats. Similar trends were observed for the conjugation rates in bile. The most abundant secondary BA in cecal content, colorectal content, and feces was hyodeoxycholic acid in WKAH rats and omega-muricholic acid in DA rats. Analyzing detailed BA profiles, including conjugation status, in a single run is possible using UPLC/ESI-MS. This method will be useful for investigating the roles of BA metabolism under physiological and pathological conditions.     

The Use of Ammonium Formate as a Mobile-Phase Modifier for LC-MS/MS Analysis of Tryptic Digests

A major challenge facing current mass spectrometry (MS)-based proteomics research is the large concentration range displayed in biological systems, which far exceeds the dynamic range of commonly available mass spectrometers. One approach to overcome this limitation is to improve online reversed-phase liquid chromatography (RP-LC) separation methodologies. LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other additives. However, TFA leads to signal suppression when incorporated with electrospray ionization (ESI), and thus, other modifiers, such as formic acid (FA), are used for LC-MS applications. FA exhibits significantly less signal suppression, but is not as effective of a modifier as TFA. An alternative mobile-phase modifier is the combination of FA and ammonium formate (AF), which has been shown to improve peptide separations. The ESI-MS compatibility of this modifier has not been investigated, particularly for proteomic applications. This work compares the separation metrics of mobile phases modified with FA and FA/AF and explores the use of FA/AF for the LC-MS analysis of tryptic digests. Standard tryptic-digest peptides were used for comparative analysis of peak capacity and sample load tolerance. The compatibility of FA/AF in proteomic applications was examined with the analysis of soluble proteins from canine prostate carcinoma tissue. Overall, the use of FA/AF improved online RP-LC separations and led to significant increases in peptide identifications with improved protein sequence coverage.     

Optimization of Data-Dependent Acquisition Parameters for Coupling High-Speed Separations with LC-MS/MS for Protein Identifications

Recent developments in chromatography, such as ultra-HPLC and superficially porous particles, offer significantly improved peptide separation. The narrow peak widths, often only several seconds, can permit a 15-min liquid chromatography run to have a similar peak capacity as a 60-min run using traditional HPLC approaches. In theory, these larger peak capacities should provide higher protein coverage and/or more protein identifications when incorporated into a proteomic workflow. We initially observed a decrease in protein coverage when implementing these faster chromatographic approaches, due to data-dependent acquisition (DDA) settings that were not properly set to match the narrow peak widths resulting from newly implemented, fast separation techniques. Oversampling of high-intensity peptides lead to low protein-sequence coverage, and tandem mass spectra (MS/MS) from lower-intensity peptides were of poor quality, as automated MS/MS events were occurring late on chromatographic peaks. These observations led us to optimize DDA settings to use these fast separations. Optimized DDA settings were applied to the analysis of Trypanosome brucei peptides, yielding peptide identifications at a rate almost five times faster than previously used methodologies. The described approach significantly improves protein identification workflows that use typical available instrumentation.     

High-sensitivity determination of tyrosine-phosphorylated peptides by on-line enzyme reactor and electrospray ionization mass spectrometry.

We describe a simple, fast, sensitive, and nonisotopic bioanalytical technique for the detection of tyrosine-phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation. The technique employs a protein tyrosine phosphatase micro enzyme reactor coupled on-line to either capillary electrophoresis or liquid chromatography and electrospray ionization mass spectrometry instruments. The micro enzyme reactor was constructed by immobilizing genetically engineered, metabolically biotinylated human protein tyrosine phosphatase beta onto the inner surface of a small piece of a 50-microns inner diameter, 360-microns outer diameter fused silica capillary or by immobilization of the phosphatase onto 40-90-microns avidin-activated resins. By coupling these reactors directly to either a capillary electrophoresis column or a liquid chromatography column, we were able to rapidly perform enzymatic dephosphorylation and separation of the reaction products. Detection and identification of the components of the reaction mixture exiting these reactors were done by mass analysis with an on-line electrospray ionization mass spectrometer. Tyrosine-phosphorylated peptides, even if present in a complex peptide mixture, were identified by subtractive analysis of peptide patterns generated with or without phosphatase treatment. Two criteria, namely a phosphatase-induced change in hydropathy and charge, respectively, and a change in molecular mass by 80 Da, were used jointly to identify phosphopeptides. We demonstrate that, with this technique, low picomole amounts of a tyrosine-phosphorylated peptide can be detected in a complex peptide mixture generated by proteolysis of a protein and that even higher sensitivities can be realized if more sensitive detection systems are applied.     

Comparative proteomic analysis of the insulin‐induced L6 myotube secretome

Emerging evidence has revealed an endocrine function for skeletal muscle; in fact, certain anti‐inflammatory cytokines are secreted only from contractile skeletal muscle. However, the skeletal muscle secretome as a whole is poorly characterized, as is how it changes in response to extracellular stimuli. Herein, we sought to identify and characterize the members of the skeletal muscle secretome, and to determine which protein secretion levels were modulated in response to insulin stimulation. To conduct these studies, we treated differentiated L6 rat skeletal muscle cells with insulin or left them untreated, and we comparatively analyzed the proteins secreted into the media. We fractionated this conditioned media using offline RP HPLC, digested the fractionated proteins, and analyzed the resulting peptides with LC‐ESI‐MS/MS. We identified a total of 254 proteins, and by using three different filtering methods, we identified 153 of these as secretory proteins. Fourteen proteins were secreted at higher levels under insulin stimulation, including several proteins known to be highly secreted in metabolic diseases; 19 proteins were secreted at lower levels under insulin stimulation. These result not only pinpointed several previously unknown, insulin induced, secretory proteins of skeletal muscle, it also described a novel approach for conditioned secretome analysis.     

Sequence analysis of peptide mixtures by automated integration of Edman and mass spectrometric data.

A computer algorithm is described that utilizes both Edman and mass spectrometric data for simultaneous determination of the amino acid sequences of several peptides in a mixture. Gas phase sequencing of a peptide mixture results in a list of observed amino acids for each cycle of Edman degradation, which by itself may not be informative and typically requires reanalysis following additional chromatographic steps. Tandem mass spectrometry, on the other hand, has a proven ability to analyze sequences of peptides present in mixtures. However, mass spectrometric data may lack a complete set of sequence-defining fragment ions, so that more than one possible sequence may account for the observed fragment ions. A combination of the two types of data reduces the ambiguity inherent in each. The algorithm first utilizes the Edman data to determine all hypothetical sequences with a calculated mass equal to the observed mass of one of the peptides present in the mixture. These sequences are then assigned figures of merit according to how well each of them accounts for the fragment ions in the tandem mass spectrum of that peptide. The program was tested on tryptic and chymotryptic peptides from hen lysozyme, and the results are compared with those of another computer program that uses only mass spectral data for peptide sequencing. In order to assess the utility of this method the program is tested using simulated mixtures of varying complexity and tandem mass spectra of varying quality.     

美洲大蠊主要变应原蛋白的质谱鉴定与分析

为了建立美洲大蠊Periplaneta americana变应原蛋白的质谱鉴定方法,我们将美洲大蠊粗浸液通过DEAE-52离子交换层析、Sephacryl S-200凝胶过滤层析等分离步骤得到纯化的74 kD蛋白,对纯化前后的该74 kD蛋白分别进行SDS-PAGE及凝胶内胰酶酶切,再经液相色谱-电喷雾-串联质谱（HPLC-ESI-MS/MS）在线联机分析,所得质谱数据进入网站（http://www.matrixscience.com）进行Mascot检索比对。通过对两者质谱鉴定结果的比较来评估美洲大蠊天然主要变应原蛋白的纯化效果。结果表明,纯化蛋白经HPLC-ESI-MS/MS鉴定是美洲大蠊主要变应原蛋白;离子交换层析等纯化步骤可以去除同一分子量的杂蛋白(如卵黄原蛋白),从而获得较好的鉴定结果。我们首次成功地运用质谱建立起变应原蛋白的新鉴定方法。     

Classification and identification of bacteria using mass spectrometry-based proteomics

Timely classification and identification of bacteria is of vital importance in many areas of public health. Mass spectrometry-based methods provide an attractive alternative to well-established microbiologic procedures. Mass spectrometry methods can be characterized by the relatively high speed of acquiring taxonomically relevant information. Gel-free mass spectrometry proteomics techniques allow for rapid fingerprinting of bacterial proteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or, for high-throughput sequencing of peptides from protease-digested cellular proteins, using mass analysis of fragments from collision-induced dissociation of peptide ions. The latter technique uses database searching of product ion mass spectra. A database contains a comprehensive list of protein sequences translated from protein-encoding open reading frames found in bacterial genomes. The results of such searches allow the assignment of experimental peptide sequences to matching theoretical bacterial proteomes. Phylogenetic profiles of sequenced peptides are then used to create a matrix of sequence-to-bacterium assignments, which are analyzed using numerical taxonomy tools. The results thereof reveal the relatedness between bacteria, and allow the taxonomic position of an investigated strain to be inferred.     

Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes

Shotgun lipidomics, comprised of intrasource separation, multidimensional mass spectrometry and computer-assisted array analysis, is an emerging powerful technique in lipidomics. Through effective intrasource separation of predetermined groups of lipid classes based on their intrinsic electrical propensities, analyses of lipids from crude extracts of biologic samples can be directly and routinely performed. Appropriate multidimensional array analysis of lipid pseudomolecular ions and fragments can be performed leading to the identification and quantitation of targeted lipid molecular species. Since most biologic lipids are linear combinations of aliphatic chains, backbones and head groups, a rich repertoire of multiple lipid building blocks present in discrete combinations represent experimental observables that can be computer reconstructed in conjunction with their pseudomolecular ions to directly determine the lipid molecular structures from a lipid extract. Through this approach, dramatic increases in the accessible dynamic range for ratiometric quantitation and discrimination of isobaric molecular species can be achieved without any prior column chromatography or operator-dependent supervision. At its current state of development, shotgun lipidomics can analyze over 20 lipid classes, hundreds of lipid molecular species and more than 95% of the mass content of a cellular lipidome. Thus, understanding the biochemical mechanisms underlying lipid-mediated disease states will be greatly facilitated by the power of shotgun lipidomics.     

Proteomic approach to aging research

The scope of the current paper is to review existing and potential applications of proteomic analysis to aging research. The focus will lie on the unique opportunities of high-throughput studies for uncovering specific alterations in protein expression, protein complexes or protein modifications caused by biological aging. The result of such studies will outline aging phenotypes and potentially indicate pathways involved in the pathogenesis of age-associated disfunctions. Specific attention is paid to the illustrations of successful applications of proteomic technologies and potential applications of new proteomic concepts to biogerontological studies.     

Identification of the hydrophobic glycoproteins of Caenorhabditis elegans

Hydrophobic proteins such as integral membrane proteins are difficult to separate, and therefore to study, at a proteomics level. However, the Asn-linked (N-linked) carbohydrates (N-glycans) contained in membrane glycoproteins are important in differentiation, embryogenesis, inflammation, cancer and metastasis, and other vital cellular processes. Thus, the identification of these proteins and their sites of glycosylation in a well-characterized model organism is the first step toward understanding the mechanisms by which N-glycans and their associated proteins function in vivo. In this report, a proteomics method recently developed by our group was applied to identify 117 hydrophobic N-glycosylated proteins of Caenorhabditis elegans extracts by analysis of 195 glycopeptides containing 199 Asn-linked oligosaccharides. Most of the proteins identified are involved in cell adhesion, metabolism, or the transport of small molecules. In addition, there are 18 proteins for which no function is known or predictable by sequence homologies and two proteins which were previously predicted to exist only on the basis of genomic sequences in the C. elegans database. Because N-glycosylation is initiated in the lumen of the endoplasmic reticulum (ER), our data can be used to reassess the previously predicted subcellular localizations of these proteins. As well, the identification of N-glycosylation sites helps establish the membrane topology of the associated glycoproteins. Caenorhabditis elegans strains are presently available with mutations in 17 of the genes we have identified. The powerful genetic tools available for C. elegans can be used to make other strains with mutations in genes encoding N-glycosylated proteins and thereby determine N-glycan function.     

A systematical analysis of tryptic peptide identification with reverse phase liquid chromatography and electrospray ion trap mass spectrometry

In this study we systematically analyzed the elution condition of tryptic peptides and the characteristics of identified peptides in reverse phase liquid chromatography and electrospray tandem mass spectrometry (RPLC-MS/MS) analysis. Following protein digestion with trypsin, the peptide mixture was analyzed by on-line RPLC-MS/MS. Bovine serum albumin (BSA) was used to optimize acetonitrile (ACN) elution gradient for tryptic peptides, and Cytochrome C was used to retest the gradient and the sensitivity of LC-MS/MS. The characteristics of identified peptides were also analyzed. In our experiments, the suitable ACN gradient is 5% to 30% for tryptic peptide elution and the sensitivity of LC-MS/MS is 50 fmol.Analysis of the tryptic peptides demonstrated that longer (more than 10 amino acids) and multi-charge state ( 2, 3) peptides are likely to be identified, and the hydropathicity of the peptides might not be related to whether it is more likely to be identified or not. The number of identified peptides for a protein might be used to estimate its loading amount under the same sample background. Moreover, in this study the identified peptides present three types of redundancy, namely identification, charge, and sequence redundancy, which may repress low abundance protein identification.     

Proteomic analysis of nuclear factors binding to an intronic enhancer in the myelin proteolipid protein gene

The myelin proteolipid protein gene ( Plp1 ) encodes the most abundant protein found in CNS myelin, accounting for nearly one-half of the total protein. Its expression in oligodendrocytes is developmentally regulated – peaking during the active myelination period of CNS development. Previously, we have identified a novel enhancer (designated ASE) in intron 1 DNA that appears to be important in mediating the surge of Plp1 gene activity during the active myelination period. Evidence suggests that the ASE participates in the formation of a specialized multi-protein/DNA complex called an enhanceosome. The current study describes an optimized, five-step, DNA affinity chromatography purification procedure to purify nuclear proteins from mouse brain that bind to the 85-bp ASE sequence, specifically. Electrophoretic mobility shift assay analysis demonstrated that specific DNA-binding activity was retained throughout the purification procedure, resulting in concomitant enrichment of nucleoprotein complexes. Identification of the purported regulatory factors was achieved through mass spectrometry analysis and included over 20 sequence-specific DNA-binding proteins. Supplementary western blot analyses to determine which of these sequence-specific factors are present in oligodendrocytes, and their developmental and regional expression in whole brain, suggest that Purα and Purβ rank highest among the candidate factors as constituents of the multi-protein complex formed on the ASE.     

Characterization of overall ceramide species in human stratum corneum

Ceramides (CERs) in human stratum corneum (SC) play physicochemical roles in determining barrier and water-holding functions of the skin, and specific species might be closely related to the regulation of keratinization, together with other CER-related lipids. Structures of those diverse CER species, however, have not been comprehensively revealed. The aim of this study was to characterize overall CER species in the SC. First, we constructed 3D multi-mass chromatograms of the overall CER species, based on normal-phase liquid chromatography (NPLC) connected to electrospray ionization-mass spectrometry (ESI-MS) using a gradient elution system and a postcolumn addition of a volatile salt-containing polar solvent. The CERs targeted from the 3D chromatograms were structurally analyzed using NPLC-ESI-tandem mass spectrometry (MS/MS), which resulted in the identification of 342 CER species in the inner forearm SC. This led to the discovery of a new CER class consisting of alpha-hydroxy fatty acid and dihydrosphingosine moieties, in addition to the 10 classes generally known. The results also revealed that those CERs contain long-chain (more than C(18))-containing sphingoids and a great number of isobaric species. These novel results will contribute not only to physiochemical research on CERs in the SC but also to lipidomics approaches to CERs in the skin.     

New tools for quantitative phosphoproteome analysis.

Recent advances in analytical methods, particularly in the area of mass spectrometry, have brought the field of proteomics to the forefront in biological science. The ultimate goal of proteomics--to characterize proteins expressed within a cell under a specific set of conditions--is daunting due to the complexity and dynamic nature the of protein population within the cell. While much of the effort has focused on developing methods to identify expressed proteins, the identification of posttranslational modifications is equally important for comprehensive proteome characterization. Of all the known posttranslational modifications, phosphorylation arguably plays the largest role in the context of cellular homeostasis. This review discusses some of the recent progress made in the development of techniques not only to identify, but also to quantitatively determine sites of phosphorylation.     

Characterization of a noncovalent lipocalin complex by liquid chromatography/electrospray ionization mass spectrometry.

Nanoscale liquid chromatography coupled to electrospray ionization mass spectrometry was used to identify the nature of the ligand that binds noncovalently to siderocalin (lipocalin 2). The folded state siderocalin-ligand complex was separated from free, unfolded siderocalin using reversed phase chromatography, and the molecular weight of the siderocalin ligand was then determined from the deconvoluted molecular weights of the complex and of the free protein. The ligand was identified as dihydroxybenzoyl-serine, a breakdown product of enterobactin, an iron-chelating compound ("siderophore") synthesized in bacteria. These results demonstrate that, in some cases, electrostatic noncovalent protein complexes can survive the denaturing conditions of reversed phase liquid chromatography and the gas phase transfer occurring during electrospray ionization.     

Proteomic analysis of two functional states of the Golgi complex in mammary epithelial cells

Organellar compartments involved in secretion are expanded during the transition from late pregnancy (basal secretory state) to lactation (maximal secretory state) to accommodate for the increased secretory function required for copious milk production in mammary epithelial cells. The Golgi complex is a major organelle of the secretory pathway and functions to sort, package, distribute, and post-translationally modify newly synthesized proteins and membrane lipids. These complex functions of the Golgi are reflected in the protein complement of the organelle. Therefore, using proteomics, the protein complements of Golgi fractions isolated at two functional states (basal and maximal) were compared to identify some of the molecular changes that occur during this transition. This global analysis has revealed that only a subset of the total proteins is up-regulated from steady state during the transition. Identification of these proteins by tandem mass spectrometry has revealed several classes of proteins involved in the regulation of membrane fusion and secretion. This first installment of the functional proteomic analysis of the Golgi complex begins to define the molecular basis for the transition from basal to maximal secretion.