Peptide mass fingerprinting by matrix-assisted laser desorption ionization mass spectrometry of proteins detected by immunostaining on nitrocellulose

We have developed an approach that allows peptide mass mapping by matrix-assisted laser desorption ionization-mass spectrometry of proteins visualized on a nitrocellulose membrane by immunochemical detection. Proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto a nitrocellulose membrane and after blocking with a nonprotein-containing polymer such as polyvinylpyrrolidone 40 (PVP-40) or Tween 20, the proteins are stained with fount India ink. After incubation with primary and, if required, secondary peroxidase-coupled antibodies, immunochemically reactive proteins can be visualized using conventional enhanced chemiluminescence detection and assigned to the India ink-stained membrane by simple superposition. The proteins of interest are excised, submitted to "on-membrane" cleavage and the peptides are analyzed by mass spectrometry. Protein-based blocking reagents normally used in standard immunodetection protocols, such as skimmed milk, can be employed. We have obtained high-quality mass spectra of bovine serum albumin (BSA) detected on an immunoblot with an estimated amount of 100 fmol applied onto the gel, indicating the sensitivity of the present method. In addition, the approach is demonstrated with two other commercially available proteins, a serum protein, the successful identification of a tyrosine phosphorylated protein from total rat liver homogenate and serine phosphorylated proteins from an EcR 293 nuclear extract separated by two-dimensional (2-D) SDS-PAGE.     

Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis

A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.     

Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: a non two-dimensional gel electrophoresis-based proteome analysis strategy

The conventional approach for analyzing the protein complement of a genome involves the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometric based protein identification technologies. While 2-DE is a powerful separation technique, it is severely limited by the insolubility of certain classes of proteins (e.g. hydrophobic membrane proteins), as well as the amount of protein that can be processed. Here, we describe a simple procedure for resolving complex mixtures of proteins that involves a combination of free flow electrophoresis (FFE), a liquid-based isoelectric focussing (IEF) method, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were identified by peptide fragment sequencing using capillary column reversed-phase high performance liquid chromatography (RP-HPLC)/mass spectrometry (MS). An initial demonstration of the method was performed using digitonin/ethylenediaminetetraacetic acid EDTA extracted cytosolic proteins from the human colon carcinoma cell line, LIM 1215. Cytosolic proteins were separated by liquid-based IEF (pH range 3-10) into 96 fractions, and each FFE fraction was further fractionated by SDS-PAGE. Selected protein bands were excised from the SDS-PAGE gel, digested in situ with trypsin, and subsequently identified by on-line RP-HPLC/electrospray-ionization ion trap MS. Our results indicate that FFE is: (i) an extremely powerful liquid-based IEF method for resolving proteins; (ii) not limited by the amount of sample that can be loaded onto the instrument; and (iii) capable of fractionating intact protein complexes (a potentially powerful tool for cell-mapping proteomics). An up-to-date list of cytosolic proteins from the human colorectal carcinoma cell line LIM 1215 can be found in the Joint Protein Structure Laboratory (JPSL) proteome database. This information will provide an invaluable resource for future proteomics-based biological studies of colon cancer. The JPSL proteome database can be accessed through the World Wide Web (WWW) network (http://www.ludwig.edu.au/jpsl/jpslhome.html).     

A screening procedure for the solubilization of chloroplast membrane proteins from the marine green macroalga Ulva lactuca using RP-HPLC-MALDI-MS

A protocol for purification and analysis of chloroplast membrane proteins in the green macroalga Ulva lactuca has been developed, including reversed phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Five different solvents were evaluated for extraction of membrane proteins by three methods. The highest protein yield was achieved when proteins were extracted directly from the chloroplasts using the solvent hexafluoroisopropanol. A range of proteins of increasing hydrophobicity was separated by HPLC. Analysis of both HPLC fractions and non-separated samples by MALDI-TOF-MS revealed proteins with molecular weights spanning between 1 and 376 kDa.     

A two-dimensional liquid-phase separation method coupled with mass spectrometry for proteomic studies of breast cancer and biomarker identification

A two-dimensional liquid-phase separation scheme coupled with mass spectrometry (MS) is presented for proteomic analysis of cell lysates from normal and malignant breast epithelial cell lines. Liquid-phase separations consist of isoelectric focusing as the first dimension and nonporous silica reverse-phase high-performance liquid chromatography (NPS-RP-HPLC) as the second dimension. Protein quantitation and mass measurement are performed using electrospray ionization-time of flight MS (ESI-TOF MS). Proteins are identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS) and MALDI-quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS). Two pH regions with 50-60 unique proteins in each pH range were chosen for analysis. Mass maps were created that allowed visualization of protein quantitation differences between normal and malignant breast epithelial cells. Of the approximately 110 unique proteins observed from mass mapping experiments over the limited pH range, 40 (36%) were positively identified by peptide mass fingerprinting and assigned to bands in the mass maps. Of these 40 proteins, 22 were more highly expressed in one or more of the malignant cell lines. These proteins represent potential breast cancer biomarkers that could aid in diagnosis, therapy, or drug development.     

Comprehensive proteome analysis of ovarian cancers using liquid phase separation, mass mapping and tandem mass spectrometry: a strategy for identification of candidate cancer biomarkers

A two-dimensional (2-D) liquid phase separation method, liquid isoelectric focusing followed by nonporous reversed-phase high performance liquid chromatography (HPLC), was used to separate proteins from human ovarian epithelial whole cell lysates. HPLC eluent was interfaced on-line to an electrospray ionization (ESI) time of flight (TOF) mass spectrometer to obtain accurate intact protein molecular weights (Mr). 2-D protein expression maps were generated displaying protein isoelectric point (pI) versus intact protein Mr. Resulting 2-D images effectively displayed quantitative differential protein expression in ovarian cancer cells versus non-neoplastic ovarian epithelial cells. Protein peak fractions were collected from the HPLC eluent, enzymatically digested, and analyzed by matrix-assisted laser desorption/ionization (MALDI) TOF-mass spectrometry (MS) peptide mass fingerprinting and by MALDI-quadrupole TOF tandem mass spectrometry peptide sequencing. Interlysate comparisons of differential protein expression between two ovarian adenocarcinoma cell lines, ES2 and MDAH-2774, and ovarian surface epithelial cells was performed. Five pI fractions from each sample were selected for comparative study and over 300 unique proteins were positively identified from the 2-D liquid expression maps using MS, which covered around 60% of proteins detected by on-line ESI-TOF-MS. This represents one of the most comprehensive proteomic analyses of ovarian cancer samples to date. Protein bands with significant up- or down-regulation in one cell line versus another as viewed in the 2-D expression maps were identified. This strategy may prove useful in identifying novel ovarian cancer marker proteins.     

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.     

High-throughput analysis of rat liver plasma membrane proteome by a nonelectrophoretic in-gel tryptic digestion coupled with mass spectrometry identification

In-gel digestion is commonly used after proteins are resolved by polyacrylamide gel electrophoresis (SDS-PAGE, 2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the direct analysis of complex proteins. Here, we describe a strategy combining isolation of purified plasma membrane, efficient digestion of plasma membrane proteins in polyacrylamide gel, and high-sensitivity analysis by advanced mass spectrometry to create a new rapid and high-throughput method. The plasma membrane protein mixture is directly incorporated into a polyacrylamide gel matrix, After formation of the gel, proteins in the gel section are digested with trypsin, and the resulting peptides are subjected to reversed-phase, high-performance liquid chromatography followed by electrospray ion-trap tandem mass analysis. Using this optimized strategy, we have identified 883 rat liver membrane proteins, of which 490 had a gene ontology (GO) annotation indicating a cellular component, and 294 (60%) of the latter were known integral membrane proteins or membrane proteins. In total, 333 proteins are predicted by the TMHMM 2.0 algorithm to have transmembrane domains (TMDs) and 52% (175 of 333) proteins to contain 2-16 TMDs. The identified membrane proteins provide a broad representation of the rat plasma membrane proteome with little bias evident due to protein p I and molecular weight (MW). Also, membrane proteins with a high GRAVY score (grand average hydrophobicity score) were identified, and basic and acidic membrane proteins were evenly represented. This study not only offered an efficient and powerful method in shotgun proteomics for the identification of proteins of complex plasma membrane samples but also allowed in-depth study of liver membrane proteomes, such as of rat models of liver-related disease. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of rat liver plasma membrane in general.     

Integration of a two-phase partition method into proteomics research on rat liver plasma membrane proteins

To comprehensively identify proteins of the rat liver plasma membrane (PM), we have adopted a proteomics strategy that utilizes sucrose density centrifugation in conjunction with aqueous two-phase partition for plasma membrane isolation, followed by SDS-PAGE, mass spectrometry and bioinformatics. Western blot analysis showed that this method results in highly purified plasma membrane fractions, which is a key to successful plasma membrane proteomics. The PM proteins were separated by SDS-PAGE and digested with trypsin. Through nano-ESI-LC MS/MS analysis we identified 428 rat liver membrane proteins, of which 304 had a gene ontology (GO) annotation indicating a cellular component, and 204 (67%) of the latter were known integral membrane proteins or membrane-associated proteins. In addition to proteins known to be associated with the plasma membrane, several hypothetical proteins have also been identified. This study not only provides a tool to study plasma membrane proteins with low levels of contamination, but also provides a data set for proteins of high to moderate abundance in rat liver plasma membranes, thus allowing for more comprehensive characterization of membrane proteins and a better understanding of membrane dynamics.     

Identification of p21Cip1 binding proteins by gel electrophoresis and capillary liquid chromatography microelectrospray tandem mass spectrometry

Proteins bound to a glutathione-S-transferase-p21Cip1 affinity column were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified using tandem mass spectrometry. Capillary liquid chromatography coupled to microelectrospray tandem mass spectrometry (capLC-microESI MS/MS) in an ion trap allowed identification of the proteins present in the gel bands. Of eleven bands analyzed, fifty-three proteins were identified. More than one hundred tryptic peptides were detected on-line, automatically fragmented and used for protein characterization in databases. Samples were also analyzed by off-line nanospray and matrix-assisted laser desorption/ionization mass spectrometry. CapLC-microESI MS/MS was the most efficient technique for the analysis of these protein mixtures.     

猪产肠毒素大肠杆菌987P蛋白受体的鉴定

产肠毒素大肠杆菌(ETEC)定植于仔猪肠道的第一步是通过987P菌毛与小肠上皮细胞表面刷状缘大分子(BBV)结合。对分离的BBV进行SDS-PAGE和Ligand blot分析表明, 在32~35KDa区域内有一条带能被987P菌毛探针所识别和结合, 所结合的条带经胰蛋白酶消化后, 通过微内径反相高效液相色谱(RP-HPLC)分离出多条主要峰带蛋白峰带, 采用衬质辅助激光解吸与电离质谱法(MALDI-MS)对主要峰带进行分析, 结合多肽氨基酸测序和Blast同源性比较, 得到3个氨基酸基序(AETAP、ALAAAGYDVEK和LGLK), 其序列与人和鼠源的组蛋白H1高度同源; 来源于仔猪小肠上皮细胞BBV的H1蛋白与BBV一样都能特异性结合纯化的987P菌毛蛋白。上述结果表明, 仔猪小肠上皮细胞BBV的组蛋白H1是987P菌毛蛋白的受体。     

猪产肠毒素大肠杆菌987P蛋白受体的鉴定

产肠毒素大肠杆菌(ETEC)定植于仔猪肠道的第一步是通过987P菌毛与小肠上皮细胞表面刷状缘大分子(BBV)结合。对分离的BBV进行SDS-PAGE和Ligand blot分析表明, 在32~35KDa区域内有一条带能被987P菌毛探针所识别和结合, 所结合的条带经胰蛋白酶消化后, 通过微内径反相高效液相色谱(RP-HPLC)分离出多条主要峰带蛋白峰带, 采用衬质辅助激光解吸与电离质谱法(MALDI-MS)对主要峰带进行分析, 结合多肽氨基酸测序和Blast同源性比较, 得到3个氨基酸基序(AETAP、ALAAAGYDVEK和LGLK), 其序列与人和鼠源的组蛋白H1高度同源; 来源于仔猪小肠上皮细胞BBV的H1蛋白与BBV一样都能特异性结合纯化的987P菌毛蛋白。上述结果表明, 仔猪小肠上皮细胞BBV的组蛋白H1是987P菌毛蛋白的受体。     

Integration of Jacobson's pellicle method into proteomic strategies for plasma membrane proteins

A modified form of the cationic colloidal silica technique for plasma membrane isolation has been combined with SDS-PAGE, mass spectrometry, and bioinformatics for evaluation as a proteomics strategy with human multiple myeloma cells and human breast cancer cells. On the basis of Western blots, half of the protein isolated is estimated to come from the plasma membrane. Forty-three percent of the 366 proteins identified by mass spectrometry had been previously classified as plasma membrane proteins. Thirty proteins previously categorized as hypothetical membrane proteins are now reported to be expressed.     

An analysis of the proteomic profile for Thermoanaerobacter tengcongensis under optimal culture conditions

The genome of Thermoanaerobacter tengcongensis is estimated to encode 2588 theoretical proteins. In this study, we have vitalized approximately 46% of the theoretical proteome experimentally using a proteomic strategy that combines three different methods, shotgun digestion plus high-performance liquid chromatography (HPLC) with ion-trap tandem mass spectrometry (shotgun-liquid chromatography (LC)/MS), one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) plus HPLC with ion-trap tandem mass spectrometry (one-dimensional electrophoresis (1DE)-LC/MS), and two-dimensional gel electrophoresis plus matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (2DE-MALDI-TOF-MS). Of the 1200 proteins identified, as few as 76 proteins were globally found by all three approaches, and notably, most of these proteins were in the soluble fraction. However, there were a number of unique proteins detected by one method only, suggesting that our strategy provides a means toward obtaining a comprehensive view of protein expression profile. Proteins from the major metabolic pathways are strongly represented on the map, and a number of these enzymes were identified by more than one proteomic method. Based upon the proteins identified in the present study, we are able to broaden the understanding of how T. tengcongensis survives under high temperature environment, whereas several of its properties can not be fully explained by genome data.     

Comprehensive proteomics in yeast using chromatographic fractionation, gas phase fractionation, protein gel electrophoresis, and isoelectric focusing

We have investigated the use of a variety of different techniques to identify as many proteins as possible in a yeast lysate, with the aim of investigating the overlap and complementarity of data from different approaches. A standard lysate was prepared from log phase yeast (Saccharomyces cerevisiae). This was then subjected to analysis via five different approaches aimed at identifying as many proteins as possible using an ion trap mass spectrometer. The total number of non-redundant protein identifications from each experiment was: 524 proteins by 2-D (SCX/C18) nanoflow liquid chromatography-liquid chromatography tandem mass spectrometry (nanoLC-LC MS/MS (MudPIT)); 381 proteins by nanoLC-MS/MS with gas phase fractionation by mass range selection; 390 proteins by nanoLC-MS/MS with gas phase fractionation by ion abundance selection; 898 proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins, in-gel digestion, and nanoLC-MS/MS of gel slices; and 422 proteins by isoelectric focusing of proteins, in-gel digestion and nanoLC-MS/MS of gel slices. The total number of non-redundant protein identifications in the five experiments was 1204. Combining only the two best experiments, the SDS-PAGE gel slices and the Mudpit, produces 1024 proteins identified, more than 85% of the total. Clearly, combining a Mudpit analysis with an SDS-PAGE gel slice experiment gives the greatest amount of protein identification information from a limited amount of sample.     

The synaptic vesicle proteome: a comparative study in membrane protein identification

A proteomic analysis of the synaptic vesicle was undertaken to obtain a better understanding of vesicle regulation. Synaptic vesicles primarily consist of integral membrane proteins that are not well resolved on traditional isoelectric focusing/two-dimensional gel electrophoresis (IEF/2-DE) gels and are resistant to in-gel digestion with trypsin thereby reducing the number of peptides available for mass spectrometric analysis. To address these limitations, two complementary 2-DE methods were investigated in the proteome analysis: (a) IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) for resolution of soluble proteins and, (b) Benzyl hexadecyl ammonium chloride/SDS-PAGE (16-BAC/SDS-PAGE) for resolution of integral membrane proteins. The IEF/SDS-PAGE method provided superior resolution of soluble proteins, but could only resolve membrane proteins with a single transmembrane domain. The 16-BAC/SDS-PAGE method improved separation, resolution and identification of integral membrane proteins with up to 12 transmembrane domains. Trypsin digestion of the integral membrane proteins was poor and fewer peptides were identified from these proteins. Analysis of both the peptide mass fingerprint and the tandem mass spectra using electrospray ionization quadrupole-time of flight mass spectrometry led to the positive identification of integral membrane proteins. Using both 2-DE separation methods, a total of 36 proteins were identified including seven integral membrane proteins, 17 vesicle regulatory proteins and four proteins whose function in vesicles is not yet known.     

Two-dimensional liquid chromatography protein expression mapping for differential proteomic analysis of normal and O157:H7 Escherichia coli

A multidimensional chromatographic method has been applied for the differential analysis of proteins from different strains of Escherichia coli bacteria. Proteins are separated in the first dimension using chromatofocusing (CF) and further separated by nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) in the second dimension. A 2-dimensional (2-D) expression map of bacterial protein content is created for virulent O157:H7 and nonvirulent E. coli strains depicting protein isoelectric point (pI) versus protein hydrophobicity. Differentially expressed proteins are further characterized using electrospray/ionization time-of-flight mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) determination and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting for protein identification. Using this method, no significant differential protein expression is exhibited between the two O157:H7 strains examined over a pH range of 4.0-7.0, and O157:H7 strains could be distinguished from nonvirulent E. coli. Several proteins differentially expressed between O157:H7 and nonvirulent E. coli are identified as potential markers for detection and treatment of O157:H7 infection.     

Sequencing of Gel-Isolated Proteins Using Microblotter Capillary Liquid Chromatography-Electrospray Mass Spectrometry

Enzymatic digests of proteins isolated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) were separated by capillary high-performance liquid chromatography (HPLC). The column eluate was split to an electrospray mass spectrometer on one side and to both a UV detector and a microblotter on the other side. Using the microblotter, the peptides eluted from the column were collected directly onto a polyvinylidene difluoride (PVDF) membrane for Edman sequencing. Thus, a peptide mass map from the mass spectrometric analysis and a prepared PVDF membrane for subsequent Edman sequencing were generated in a single experiment. The addition of molecular mass information to the blotted LC eluate is useful for determining the most important peaks to undergo Edman sequencing. Coupling the capillary HPLC with a microblotter to electrospray mass spectrometry provides an integrated system for separation, collection, and structural analysis of protein digests. It provides high levels of sensitivity, recovery, and convenience for protein characterization. Proteins loaded onto SDS–PAGE at low picomole levels can be analyzed by the new integrated system.     

Identification of Intact High Molecular Weight Glutenin Subunits from the Wheat Proteome Using Combined Liquid Chromatography-Electrospray Ionization Mass Spectrometry

The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC), and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for further top-down proteomics of individual HMW-GS and the entire wheat glutenin fraction.