Human plasma proteome analysis by multidimensional chromatography prefractionation and linear ion trap mass spectrometry identification

A resurgence of interest in the human plasma proteome has occurred in recent years because it holds great promise of revolution in disease diagnosis and therapeutic monitoring. As one of the most powerful separation techniques, multidimensional liquid chromatography has attracted extensive attention, but most published works have focused on the fractionation of tryptic peptides. In this study, proteins from human plasma were prefractionated by online sequential strong cation exchange chromatography and reversed-phase chromatography. The resulting 30 samples were individually digested by trypsin, and analyzed by capillary reversed-phase liquid chromatography coupled with linear ion trap mass spectrometry. After meeting stringent criteria, a total of 1292 distinct proteins were successfully identified in our work, among which, some proteins known to be present in serum in <10 ng/mL were detected. Compared with other works in published literatures, this analysis offered a more full-scale list of the plasma proteome. Considering our strategy allows high throughput of protein identification in serum, the prefractionation of proteins before MS analysis is a simple and effective method to facilitate human plasma proteome research.     

The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins

Vacuoles play central roles in plant growth, development, and stress responses. To better understand vacuole function and biogenesis we have characterized the vegetative vacuolar proteome from Arabidopsis thaliana. Vacuoles were isolated from protoplasts derived from rosette leaf tissue. Total purified vacuolar proteins were then subjected either to multidimensional liquid chromatography/tandem mass spectrometry or to one-dimensional SDS-PAGE coupled with nano-liquid chromatography/tandem mass spectrometry (nano-LC MS/MS). To ensure maximum coverage of the proteome, a tonoplast-enriched fraction was also analyzed separately by one-dimensional SDS-PAGE followed by nano-LC MS/MS. Cumulatively, 402 proteins were identified. The sensitivity of our analyses is indicated by the high coverage of membrane proteins. Eleven of the twelve known vacuolar-ATPase subunits were identified. Here, we present evidence of four tonoplast-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), representing each of the four groups of SNARE proteins necessary for membrane fusion. In addition, potential cargo of the N- and C-terminal propeptide sorting pathways, association of the vacuole with the cytoskeleton, and the vacuolar localization of 89 proteins of unknown function are identified. A detailed analysis of these proteins and their roles in vacuole function and biogenesis is presented.     

Comparison of strong cation exchange and SDS-PAGE fractionation for analysis of multiprotein complexes

Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. Although affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.     

The proteome of the bacterium Mycoplasma pneumoniae: comparing predicted open reading frames to identified gene products

An existing proteome map of the bacterium Mycoplasma pneumoniae comprising proteins from 224 genes was extended to 305 genes. This corresponds to about 44% of the 688 proposed genome sequence derived open reading frames (ORFs). The newly assigned gene products were enriched, separated by one-dimensional or two-dimensional (2-D) gel electrophoresis and identified by mass spectrometry. The enrichment procedures included differential centrifugation, anion and cation exchange chromatography, affinity chromatography with heparin as a ligand and isolation of biotinylated proteins by binding to immobilized streptavidin. A comparative analysis of the identified proteins from 305 genes with the as yet unverified 383 ORFs concerning isoelectric point, molecular weight and number of transmembrane segments revealed that proteins with more than three predicted transmembrane segments and an isoelectric point above 10.5 are most likely not to be separated by 2-D gel electrophoresis. The mutual benefits of genomics and proteomics were shown by the identification of a todate unannotated 128 amino acid long protein.     

Characterization of the human blood plasma proteome

We describe methods for broad characterization of the human plasma proteome. The combination of stepwise immunoglobulin G (IgG) and albumin protein depletion by affinity chromatography and ultrahigh-efficiency capillary liquid chromatography separations coupled to ion trap-tandem mass spectrometry enabled identification of 2392 proteins from a single plasma sample with an estimated confidence level of > 94%, and an additional 2198 proteins with an estimated confidence level of 80%. The relative abundances of the identified proteins span a range of over eight orders of magnitude in concentration (< 30 pg/mL to approximately 30 mg/mL), facilitated by the attomole-level sensitivity of the analysis methods. More than 80% of the observed proteins demonstrate interactions with IgG and/or albumin, and the human plasma protein loss in the affinity chromatography/strong cation exchange/reversed-phase liquid chromatography-tandem mass spectrometry methodology was investigated in detail. The results of this study provide a basis for a wide range of plasma proteomics studies, including broad quantitation of relative abundances in comparative studies of the identification of novel protein disease markers, as well as further studies of protein-protein interactions.     

Analysis of mouse liver membrane proteins using multidimensional separations and tandem mass spectrometry

In the field of proteomic investigation, the analysis of membrane proteins still faces many technical challenges. A fundamental question in this puzzle is how to maintain a proper solvent environment to allow the hydrophobic proteins to remain solubilized. We propose that the denaturation of membrane proteins in a highly concentrated urea solution enables them to be ionized such that ionic exchange chromatography can be employed to separate them. The membrane proteins prepared from the mouse liver were dissolved in 6M guanidine hydrochloride, 20mM Tris-HCl, pH 9.0, and loaded onto a tandem chromatography apparatus coupled with Q-Sepharose FF and Sephacryl S-200HR. These columns were able to adsorb 97.87% of the membrane protein preparations. Using a linear NaCl (0-1.0M) gradient, the bound proteins were eluted out at 0.1-1.0M NaCl, and examined by SDS-PAGE. Furthermore the protein bands underwent excision and digestion with trypsin, followed by reverse-phase chromatography for the separation of the digested peptides and ionic-trap mass spectrometry for the identification of the proteins. From the SDS-PAGE gels, the overlap between proteins from neighboring bands was only 21.34%, indicating that the anionic-size exclusion coupling chromatography efficiently separated these membrane proteins. Of a total of 392 proteins identified, 306 were membrane proteins or membrane-associated proteins. Based on the calculation of hydrophobicity, the GRAVY scores of 83 proteins are greater than, or equal to, 0.00. Taking all of this evidence together, our results revealed that this approach is satisfactory for studies on the membrane proteome from the mouse liver.     

Characterization of the low molecular weight human serum proteome

Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome.     

Lumbar cerebrospinal fluid proteome in multiple sclerosis: characterization by ultrafiltration, liquid chromatography, and mass spectrometry

Neurological diseases, including multiple sclerosis (M.S.), often provoke changes in the functioning of the endothelial and epithelial brain barriers and give rise to disease-associated alterations of the cerebrospinal fluid (CSF) proteome. In the present study, pooled and ultrafiltered CSF of M.S. and non-M.S. patients was digested with trypsin and analyzed by off-line strong cation-exchange chromatography (SCX) coupled to on-line reversed-phase LC-ESI-MS/MS. In an alternative approach, the trypsin-treated subproteomes were analyzed directly by LC-ESI-MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both proteomic approaches in combination with a three-step evaluation process including the search engines Sequest and Mascot, and the validation software Scaffold, resulted in the identification of 148 proteins. Sixty proteins were identified in CSF for the first time by mass spectrometry. For validation purposes, the concentration of cystatin A was determined in individual CSF and serum samples of M.S. and non-M.S. patients using ELISA.     

Peptide mass fingerprinting

Peptide mass fingerprinting by MALDI-MS and sequencing by tandem mass spectrometry have evolved into the major methods for identification of proteins following separation by two-dimensional gel electrophoresis, SDS-PAGE or liquid chromatography. One main technological goal of proteome analyses beside high sensitivity and automation was the comprehensive analysis of proteins. Therefore, the protein species level with the essential information on co- and post-translational modifications must be achieved. The power of peptide mass fingerprinting for protein identification was described here, as exemplified by the identification of protein species with high molecular masses (spectrin alpha and beta), low molecular masses (elongation factor EF-TU fragments), splice variants (alpha A crystallin), aggregates with disulfide bridges (alkylhydroperoxide reductase), and phosphorylated proteins (heat shock protein 27). Helpful tools for these analyses were the use of the minimal protein identifier concept and the software program MS-Screener to remove mass peaks assignable to contaminants and neighbor spots.     

Evaluation of a solution isoelectric focusing protocol as an alternative to ion exchange chromatography for charge-based proteome prefractionation

Solution isoelectric focusing (sIEF) is evaluated relative to ion exchange chromatography (IEC) as a preferred charge-based prefractionation tool for proteome mixtures. While IEC is extensively employed for proteome prefractionation prior to MS analysis, we demonstrate here that conventional salt gradient IEC has significant shortcomings compared to sIEF. Here, we critically evaluated a custom eight-channel sIEF device for intact protein separation, relative to strong cation exchange (SCX) and strong anion exchange (SAX) chromatography. The resolution, recovery, and uniformity of separation obtained with our sIEF device were comparable or superior to that of optimized IEC separations. Most importantly for intact proteins, sIEF separations strongly correlate with the proteins’ isoelectric point, which contrasts with IEC where no correlation was observed. To validate the sIEF platform for proteome analysis, prefractionation through sIEF resulted in the confident identification of a greater number of proteins from yeast (211) following LC–MS/MS, relative to those obtained through SAX (173) or SCX (148).     

A proteomic view of Bifidobacterium infantis generated by multi-dimensional chromatography coupled with tandem mass spectrometry

Bifidobacteria are Gram-positive prokaryotes that naturally colonize the human gut where they exert several health-promoting effects. The present paper reports the use of a strong cation exchange-reversed-phase-tandem mass spectrometry strategy to catalogue the most abundantly expressed proteins of a probiotic Bifidobacterium infantis strain. A global view of the B. infantis proteome was obtained. The bimodal representation of the proteins identified by mass spectrometry provides the first theoretical two-dimensional map of protein distribution for this organism. Among the 136 proteins identified by multidimensional protein identification technology (MudPIT) analysis, 118 showed the highest similarity with the translated sequences of B. longum genome, two proteins were similar to other Bifidobacterium species and the remaining 16 were similar to different genera. Specific biological activities have been assigned to 115 identified proteins, whereas 21 have been referred to the group of hypothetical proteins. The MudPIT approach allowed us to identify high mass and basic isoelectric point proteins that are generally challenging to visualize using the traditional two-dimensional electrophoresis technique. Redundancy in peptide and protein identification using the double chromatography technique was also evaluated.     

Simplified proteomics approach to discover protein-ligand interactions

Identifying targets of biologically active small molecules is an essential but still challenging task in drug research and chemical genetics. Energetics-based target identification is an approach that utilizes the change in the conformational stabilities of proteins upon ligand binding in order to identify target proteins. Different from traditional affinity-based capture approaches, energetics-based methods do not require any labeling or immobilization of the test molecule. Here, we report a surprisingly simple version of energetics-based target identification, which only requires ion exchange chromatography, SDS PAGE, and minimal use of mass spectrometry. The complexity of a proteome is reduced through fractionation by ion exchange chromatography. Urea-induced unfolding of proteins in each fraction is then monitored by the significant increase in proteolytic susceptibility upon unfolding in the presence and the absence of a ligand. Proteins showing a different degree of unfolding with the ligand are identified by SDS PAGE followed by mass spectrometry. Using this approach, we identified ATP-binding proteins in the Escherichia coli proteome. In addition to known ATP-binding proteins, we also identified a number of proteins that were not previously known to interact with ATP. To validate one such finding, we cloned and purified phosphoglyceromutase, which was not previously known to bind ATP, and confirmed that ATP indeed stabilizes this protein. The combination of fractionation and pulse proteolysis offers an opportunity to investigate protein-drug or protein-metabolite interactions on a proteomic scale with minimal instrumentation and without modification of a molecule of interest.     

Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes

In-depth analysis of the serum and plasma proteomes by mass spectrometry is challenged by the vast dynamic range of protein abundance and substantial complexity. There is merit in reducing complexity through fractionation to facilitate mass spectrometry analysis of low-abundance proteins. However, fractionation reduces throughput and has the potential of diluting individual proteins or inducing their loss. Here, we have investigated the contribution of extensive fractionation of intact proteins to depth of analysis. Pooled serum depleted of abundant proteins was fractionated by an orthogonal two-dimensional system consisting of anion-exchange and reversed-phase chromatography. The resulting protein fractions were aliquotted; one aliquot was analyzed by shotgun LC-MS/MS, and another was further resolved into protein bands in a third dimension using SDS-PAGE. Individual gel bands were excised and subjected to in situ digestion and mass spectrometry. We demonstrate that increased fractionation results in increased depth of analysis based on total number of proteins identified in serum and based on representation in individual fractions of specific proteins identified in gel bands following a third-dimension SDS gel analysis. An intact protein analysis system (IPAS) based on a two-dimensional plasma fractionation schema was implemented that resulted in identification of 1662 proteins with high confidence with representation of protein isoforms that differed in their chromatographic mobility. Further increase in depth of analysis was accomplished by repeat analysis of aliquots from the same set of two-dimensional fractions resulting in overall identification of 2254 proteins. We conclude that substantial depth of analysis of proteins from milliliter quantities of serum or plasma and detection of isoforms are achieved with depletion of abundant proteins followed by two-dimensional protein fractionation and MS analysis of individual fractions.     

Genome-wide proteomics of Natronomonas pharaonis

The aerobic, haloalkaliphilic archaeon Natronomonas pharaonis is able to survive in salt-saturated lakes of pH 11. According to genome analysis, the theoretical proteome consists of 2843 proteins. To reach further conclusions about its cellular physiology, the cytosolic protein inventory of Nmn. pharaonis has been analyzed using MS/MS on an ESI-Q-TOF mass spectrometer coupled on-line with a nanoLC system. The efficiency of this shotgun approach is illustrated by the identification of 929 proteins of which 886 are soluble proteins representing 41% of the cytosolic proteome. Cell lysis under denaturing conditions in water with subsequent separation by SDS-PAGE prior to nanoLC-MS/MS resulted in identification of 700 proteins. The same number (but a different subset) of proteins was identified upon cell lysis under native conditions followed by size fractionation (retaining protein complexes) prior to SDS-PAGE. Additional size fractionation reduced sample complexity and increased identification reliability. The set of identified proteins covers about 60% of the cytosolic proteins involved in metabolism and genetic information processing. Many of the identified proteins illustrate the high genetic variability among the halophilic archaea.     

Proteomics identification of proteins in human cortex using multidimensional separations and MALDI tandem mass spectrometer

It is essential to characterize the proteome of various regions of human brain because most, if not all, neurodegenerative diseases are region-specific. Here we report an in-depth proteomics identification of proteins extracted from the frontal cortex, a region playing a critical role in cognitive function. The integrated proteomics analytical flow consisted of biochemical fractionation, strong cation exchange chromatography, reverse phase liquid chromatography, and MALDI-TOF/TOF mass spectrometric analysis. In total, 812 proteins were confidently identified with two or more peptides. These proteins demonstrated diverse isoelectric points and molecular weights and are involved in several molecular functions, including protein binding, catalytic activity, transport, structure, and signal transduction. A number of proteins known to be associated with neurodegenerative diseases were also identified. Detailed characterization of these proteins will supply the necessary information to appropriately interpret proteins associated with aging and/or age-related neurodegenerative diseases. Finally 140 proteins found in the cortical proteome were present in the proteome of cerebrospinal fluid, providing tissue-specific candidates for biomarker discovery in body fluid.     

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.     

The human platelet membrane proteome reveals several new potential membrane proteins

We present the first focused proteome study on human platelet membranes. Due to the removal of highly abundant cytoskeletal proteins a wide spectrum of known platelet membrane proteins and several new and hypothetical proteins were accessible. In contrast to other proteome studies we focused on prefractionation and purification of membranes from human platelets according to published protocols to reduce sample complexity and enrich interesting membrane proteins. Subsequently protein separation by common one-dimensional SDS-PAGE as well as the combined benzyldimethyl-n-hexadecylammonium chloride/SDS separation technique was performed prior to mass spectrometry analysis by nano-LC-ESI-MS/MS. We demonstrate that the application of both separation systems in parallel is required for maximization of protein tagging out of a complex sample. Furthermore the identification of several potential membrane proteins in human platelets yields new potential targets in functional platelet research.     

Proteomic analysis of human neutrophil granules

Stimulated exocytosis of intracellular granules plays a critical role in conversion of inactive, circulating neutrophils to fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing. The functional changes induced by exocytosis of each of the granule subsets, gelatinase (tertiary) granules, specific (secondary) granules, and azurophil (primary) granules, are poorly defined. To improve the understanding of the role of exocytosis of these granule subsets, a proteomic analysis of the azurophil, specific, and gelatinase granules from human neutrophils was performed. Two different methods for granule protein identification were applied. First, two-dimensional (2D) gel electrophoresis followed by MALDI-TOF MS analysis of peptides obtained by in-gel trypsin digestion of proteins was performed. Second, peptides from tryptic digests of granule membrane proteins were separated by two-dimensional microcapillary chromatography using strong cation exchange and reverse phase microcapillary high pressure liquid chromatography and analyzed with electrospray ionization tandem mass spectrometry (2D HLPC ESI-MS/MS). Our analysis identified 286 proteins on the three granule subsets, 87 of which were identified by MALDI MS and 247 were identified by 2D HPLC ESI-MS/MS. The increased sensitivity of 2D HPLC ESI-MS/MS, however, resulted in identification of over 500 proteins from subcellular organelles contaminating isolated granules. Defining the proteome of neutrophil granule subsets provides a basis for understanding the role of exocytosis in neutrophil biology. Additionally, the described methods may be applied to mobilizable compartments of other secretory cells.     

Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry

Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poor specificity. We investigated the potential of IMAC in combination with capillary liquid chromatography coupled to tandem mass spectrometry for the identification of plasma membrane phosphoproteins of Arabidopsis. Without chemical modification of peptides, over 75% pure phosphopeptides were isolated from plasma membrane digests and detected and sequenced by mass spectrometry. We present a scheme for two-dimensional peptide separation using strong anion exchange chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage of monophosphorylated peptides. Among the identified sequences, six originated from different isoforms of the plasma membrane H(+)-ATPase and defined two previously unknown phosphorylation sites at the regulatory C terminus. The potential for large-scale identification of phosphorylation sites on plasma membrane proteins will have wide-ranging implications for research in signal transduction, cell-cell communication, and membrane transport processes.