Proteome analysis of Escherichia coli using high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry

The basic problem of complexity poses a significant challenge for proteomic studies. To date two-dimensional gel electrophoresis (2-DE) followed by enzymatic in-gel digestion of the peptides, and subsequent identification by mass spectrometry (MS) is the most commonly used method to analyze complex protein mixtures. However, 2-DE is a slow and labor-intensive technique, which is not able to resolve all proteins of a proteome. To overcome these limitations gel-free approaches are developed based on high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The high resolution and excellent mass accuracy of FT-ICR MS provides a basis for simultaneous analysis of numerous compounds. In the present study, a small protein subfraction of an Escherichia coli cell lysate was prepared by size-exclusion chromatography and proteins were analyzed using C4 reversed phase (RP)-HPLC for pre-separation followed by C18 RP nanoHPLC/nanoESI FT-ICR MS for analysis of the peptide mixtures after tryptic digestion of the protein fractions. We identified 231 proteins and thus demonstrated that a combination of two RP separation steps - one on the protein and one on the peptide level - in combination with high-resolution FT-ICR MS has the potential to become a powerful method for global proteomics studies.     

A proteomic analysis of human cilia: identification of novel components

Cilia play an essential role in protecting the respiratory tract by providing the force necessary for mucociliary clearance. Although the major structural components of human cilia have been described, a complete understanding of cilia function and regulation will require identification and characterization of all ciliary components. Estimates from studies of Chlamydomonas flagella predict that an axoneme contains > or = 250 proteins. To identify all the components of human cilia, we have begun a comprehensive proteomic analysis of isolated ciliary axonemes. Analysis by two-dimensional (2-D) PAGE resulted in a highly reproducible 2-D map consisting of over 240 well resolved components. Individual protein spots were digested with trypsin and sequenced using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Peptide matches were obtained to 38 potential ciliary proteins by this approach. To identify ciliary components not resolved by 2-D PAGE, axonemal proteins were separated on a one-dimensional gel. The gel lane was divided into 45 individual slices, each of which was analyzed by LC/MS/MS. This experiment resulted in peptide matches to an additional 110 proteins. In a third approach, preparations of isolated axonemes were digested with Lys-C, and the resulting peptides were analyzed directly by LC/MS/MS or by multidimensional LC/MS/MS, leading to the identification of a further 66 proteins. Each of the four approaches resulted in the identification of a subset of the proteins present. In total, sequence data were obtained on over 1400 peptides, and over 200 potential axonemal proteins were identified. Peptide matches were also obtained to over 200 human expressed sequence tags. As an approach to validate the mass spectrometry results, additional studies examined the expression of several identified proteins (annexin I, sperm protein Sp17, retinitis pigmentosa protein RP1) in cilia or ciliated cells. These studies represent the first proteomic analysis of the human ciliary axoneme and have identified many potentially novel components of this complex organelle.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Protein identification assisted by the prediction of retention time in liquid chromatography/tandem mass spectrometry

Two-dimensional liquid chromatography (2D-LC) coupled on-line with electrospray ionization tandem mass spectrometry (2D-LC-ESI-MS/MS) is a new platform for analysis and identification of proteome. Peptides are separated by 2D-LC and then performed MS/MS analysis by tandem MS/MS. The MS/MS data are searched against database for protein identification. In one 2D-LC-ESI-MS/MS run, we obtained not only the structural information of peptides directly from MS/MS, but also the retention time of peptides eluted from LC. Information on the chromatographic behavior of peptides can assist protein identification in the new platform for proteomics. The retention time of the matching peptides of the identified protein was predicted by the hydrophobic contribute of each amino acid on reversed-phase liquid chromatography (RPLC). By using this strategy proteins were identified by four types of information: peptide mass fingerprinting (PMF), sequence query, and MS/MS ions searched and the predicted retention time. This additional information obtained from LC could assist protein identification with no extra experimental cost.     

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.     

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.     

Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome

Highly complex protein mixtures can be directly analyzed after proteolysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this paper, we have utilized the combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS. One milligram of whole yeast protein was proteolyzed and separated by SCX chromatography (2.1 mm i.d.) with fraction collection every minute during an 80-min elution. Eighty fractions were reduced in volume and then re-injected via an autosampler in an automated fashion using a vented-column (100 microm i.d.) approach for RP-LC-MS/MS analysis. More than 162,000 MS/MS spectra were collected with 26,815 matched to yeast peptides (7,537 unique peptides). A total of 1,504 yeast proteins were unambiguously identified in this single analysis. We present a comparison of this experiment with a previously published yeast proteome analysis by Yates and colleagues (Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-7). In addition, we report an in-depth analysis of the false-positive rates associated with peptide identification using the Sequest algorithm and a reversed yeast protein database. New criteria are proposed to decrease false-positives to less than 1% and to greatly reduce the need for manual interpretation while permitting more proteins to be identified.     

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.     

Membrane protein identifications by mass spectrometry using electrocapture-based separation as part of a two-dimensional fractionation system

A two-dimensional (2D) separation method was used to decrease sample complexity in analysis of tryptic peptides from glomerular membrane proteins by tandem mass spectrometry (MS/MS). The first dimension was carried out by electrocapture (EC), which fractionates peptides according to electrophoretic mobility. The second dimension was reverse-phase liquid chromatography (RP-LC), in which EC fractions were further separated and analyzed online by MS/MS. Using this methodology, we now identify 102 glomerular proteins (57 membrane proteins). Many peptides were possible to observe and select for MS/MS only using the 2D approach. Others were detectable in both one-dimensional (1D, without the EC step) and 2D experiments but were selectable for sequence analysis only from the 2D separations because the decrease in complexity then gives time for the mass analyzer to select the peptide and switch to the MS/MS mode. A minority of the peptides were detectable only in the 1D mode (presumably because of handling losses), but at the end this did not decrease the number of proteins identified by the 2D separation. After a database search, the combination of EC and RP-LC MS/MS versus a 1D RP-LC MS/MS separation resulted in a threefold increase in the number of proteins identified and improved the sequence coverage in the identifications, bringing our proteome-identified glomerular proteins to 282.     

Protein phosphorylation and expression profiling by Yin-yang multidimensional liquid chromatography (Yin-yang MDLC) mass spectrometry

A system which consisted of multidimensional liquid chromatography (Yin-yang MDLC) coupled with mass spectrometry was used for the identification of peptides and phosphopeptides. The multidimensional liquid chromatography combines the strong-cation exchange (SCX), strong-anion exchange (SAX), and reverse-phase methods for the separation. Protein digests were first loaded on an SCX column. The flow-through peptides from SCX were collected and further loaded on an SAX column. Both columns were eluted by offline pH steps, and the collected fractions were identified by reverse-phase liquid chromatography tandem mass spectrometry. Comprehensive peptide identification was achieved by the Yin-yang MDLC-MS/MS for a 1 mg mouse liver. In total, 14 105 unique peptides were identified with high confidence, including 13 256 unmodified peptides and 849 phosphopeptides with 809 phosphorylated sites. The SCX and SAX in the Yin-Yang system displayed complementary features of binding and separation for peptides. When coupled with reverse-phase liquid chromatography mass spectrometry, the SAX-based method can detect more extremely acidic (pI < 4.0) and phosphorylated peptides, while the SCX-based method detects more relatively basic peptides (pI > 4.0). In total, 134 groups of phosphorylated peptide isoforms were obtained, with common peptide sequences but different phosphorylated states. This unbiased profiling of protein expression and phosphorylation provides a powerful approach to probe protein dynamics, without using any prefractionation and chemical derivation.     

A dataset of human fetal liver proteome identified by subcellular fractionation and multiple protein separation and identification technology

A high throughput process including subcellular fractionation and multiple protein separation and identification technology allowed us to establish the protein expression profile of human fetal liver, which was composed of at least 2,495 distinct proteins and 568 non-isoform groups identified from 64,960 peptides and 24,454 distinct peptides. In addition to the basic protein identification mentioned above, the MS data were used for complementary identification and novel protein mining. By doing the analysis with integrated protein, expressed sequence tag, and genome datasets, 223 proteins and 15 peptides were complementarily identified with high quality MS/MS data.     

High-sensitivity analysis of human plasma proteome by immobilized isoelectric focusing fractionation coupled to mass spectrometry identification

Immobilized pH gradients isoelectric focusing (IPG-IEF) is the first dimension typically used in two-dimensional gel electrophoresis (2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the analysis of proteins. Here, we described a strategy combining isoelectric focusing in immobilized pH gradient strips, and mass spectrometry to create a new high-throughput and sensitive detection method. Protein mixture is separated by in-gel IEF, then the entire strip is cut into a set of gel sections. Proteins in each gel section are digested with trypsin, and the resulted peptides are subjected to reversed-phase high performance liquid chromatography followed by electrospray-linear ion-trap tandem mass analysis. Using this optimized strategy, we have identified 744 distinct human proteins from an IPG strip loaded only 300 microg of plasma proteins. When compared with other works in published literatures, this study offered a more convenient and sensitive method from gel to mass spectrometry for the separation and identification proteins of complex biological samples.     

Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins

Prefractionations of proteins prior to their proteolysis, chromatography, and MS/MS analyses help reduce complexity and increase the yield of protein identifications. A number of methods were evaluated here for prefractionating serum samples distributed to the participating laboratories as part of the human Plasma Proteome Project. These methods include strong cation exchange (SCX) chromatography, slicing of SDS-PAGE gel bands, and liquid-phase IEF of the proteins. The fractionated proteins were trypsinized and the resulting peptides were resolved and analyzed by multidimensional protein identification technology coupled to IT MS/MS. The MS/MS spectra were clustered, combined, and searched against the IPI protein databank using Pep-Miner. The identification results were evaluated for the efficacy of the different prefractionation methodologies to identify larger numbers of proteins at higher confidence and to achieve the best coverage of the proteins with the identified peptides. Prefractionation based on SCX resulted in the largest number of identified proteins, followed by gel slices and then the liquid-phase IEF. An important observation was that each of the methods revealed a set of unique proteins, some identified with high confidence. Therefore, for comprehensive identification of the serum proteins, several different prefractionation approaches should be used in parallel.     

Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin

A proteomics method has been developed to purify and identify the specific proteins modified by ubiquitin (Ub) from human cells. In purified samples, Ub and 21 other proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra using SEQUEST. These proteins included several of the expected carriers of Ub including Ub-conjugating enzymes and histone proteins. To perform these experiments, a cell line coexpressing epitope tagged His(6X)-Ub and green fluorescent protein (GFP) was generated by stably transfecting HEK293 cells. Ubiquitinated proteins were purified using nickel-affinity chromatography and digested in solution with trypsin. Complex mixtures of peptides were separated by reversed phase chromatography and analyzed by nano LC-MS/MS using the LCQ quadrupole ion-trap mass spectrometer. Proteins identified from His(6X)-Ub-GFP transfected cells were compared to a list of proteins from HEK293 cells, which associate with nickel-nitrilotriacetic acid (Ni-NTA)-agarose in the absence of His-tagged Ub. In a proof of principle experiment, His(6X)-Ub-GFP transfected cells were treated with As (III) (10 microM, 24 h) in an attempt to identify substrates increasingly modified by Ub. In this experiment, proliferating cell nuclear antigen, a DNA repair protein and known ubiquitin substrate, was confidently identified. This proteomics method, developed for the analysis of ubiquitinated proteins, is a step towards large-scale characterization of Ub-protein conjugates in numerous physiological and pathological states.     

In silico analysis of accurate proteomics, complemented by selective isolation of peptides

Protein identification by mass spectrometry is mainly based on MS/MS spectra and the accuracy of molecular mass determination. However, the high complexity and dynamic ranges for any species of proteomic samples, surpass the separation capacity and detection power of the most advanced multidimensional liquid chromatographs and mass spectrometers. Only a tiny portion of signals is selected for MS/MS experiments and a still considerable number of them do not provide reliable peptide identification. In this article, an in silico analysis for a novel methodology of peptides and proteins identification is described. The approach is based on mass accuracy, isoelectric point (pI), retention time (t(R)) and N-terminal amino acid determination as protein identification criteria regardless of high quality MS/MS spectra. When the methodology was combined with the selective isolation methods, the number of unique peptides and identified proteins increases. Finally, to demonstrate the feasibility of the methodology, an OFFGEL-LC-MS/MS experiment was also implemented. We compared the more reliable peptide identified with MS/MS information, and peptide identified with three experimental features (pI, t(R), molecular mass). Also, two theoretical assumptions from MS/MS identification (selective isolation of peptides and N-terminal amino acid) were analyzed. Our results show that using the information provided by these features and selective isolation methods we could found the 93% of the high confidence protein identified by MS/MS with false-positive rate lower than 5%.     

Identification of the human beta-casein C-terminal fragments that specifically bind to purified antibodies to bovine beta-lactoglobulin

The presence of foreign proteins in human milk after the ingestion of bovine dairy products is thought to be one of the possible causes of allergic sensitization in exclusively breast-fed predisposed infants. The immunologic determination of bovine beta-lactoglobulin (LG) concentration in human milk has been reported by several researchers, but the results are conflicting. Moreover, a strong cross-reactivity between antibodies to bovine beta-LG and human milk proteins and peptides was reported, throwing doubt on the reliability of radioimmunoassay and enzyme-linked immunosorbent assay detection and quantification assays for bovine beta-LG in human milk. Thus, the goal of this study was to isolate human milk peptides with a molecular mass >or= 1,000 Da cross-reactive with antibodies to bovine beta-LG in order to identify possible common epitopes between human and bovine milk proteins. The proteins were first isolated by affinity chromatography with purified polyclonal antibodies to bovine beta-LG, followed by gel filtration fast phase liquid chromatography and reverse phase-high performance liquid chromatography purification of the components specifically bound in the affinity separation step. Affinity-bound peptides were identified by determining their amino acid sequence. All the sequenced peptides belonged to the C-terminal part of human beta-casein, which confirms the cross-reactivity of human milk proteins and peptides with antibodies to bovine beta-LG and allows the identification of possible common epitopes between the two proteins. No bovine beta-LG peptides with a molecular mass >or= 1,000 Da were found in our milk samples from healthy mothers on a diet rich in bovine milk and dairy products.     

Global phosphoproteome of HT-29 human colon adenocarcinoma cells

Phosphorylation events in cellular signaling cascades triggered by a variety of cellular stimuli modulate protein function, leading to diverse cellular outcomes including cell division, growth, death, and differentiation. Abnormal regulation of protein phosphorylation due to mutation or overexpression of signaling proteins often results in various disease states. We provide here a list of protein phosphorylation sites identified from HT-29 human colon adenocarcinoma cell line by immobilized metal affinity chromatography (IMAC) combined with liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis. In this study, proteins extracted from HT-29 whole cell lysates were digested with trypsin and carboxylate groups on the resulting peptides were converted to methyl esters. Derivatized phosphorylated peptides were enriched using Fe(3+)-chelated metal affinity resin. Phosphopeptides retained by IMAC were separated by high performance liquid chromatography (HPLC) and analyzed by electrospray ionization-quadrupole-time-of-flight (ESI-Q-TOF) mass spectrometry. We identified 238 phosphorylation sites, 213 of which could be conclusively localized to a single residue, from 116 proteins by searching MS/MS spectra against the human protein database using MASCOT. Peptide identification and phosphorylation site assignment were confirmed by manual inspection of the MS/MS spectra. Many of the phosphorylation sites identified in our results have not been described previously in the scientific literature. We attempted to ascribe functionality to the sites identified in this work by searching for potential kinase motifs with Scansite (http://scansite.mit.edu) and obtaining information on kinase substrate selectivity from Pattern Explorer (http://scansite.mit.edu/pe). The list of protein phosphorylation sites identified in the present experiment provides broad information on phosphorylated proteins under normal (asynchronous) cell culture conditions. Sites identified in this study may be utilized as surrogate bio-markers to assess the activity of selected kinases and signaling pathways from different cell states and exogenous stimuli.     

High recovery HPLC separation of lipid rafts for membrane proteome analysis

Proteomic analysis of complex samples can be facilitated by protein fractionation prior to enzymatic or chemical fragmentation combined with MS-based identification of peptides. Although aqueous soluble protein fractionation by liquid chromatography is relatively straightforward, membrane protein separations have a variety of technical challenges. Reversed-phase high performance liquid chromatography (RP-HPLC) separations of membrane proteins often exhibit poor recovery and bandwidths, and generally require extensive pretreatment to remove lipids and other membrane components. Human brain tissue lipid raft protein preparations have been used as a model system to develop RP-HPLC conditions that are effective for protein fractionation, and are compatible with downstream proteomic analytical workflows. By the use of an appropriate RP column material and operational conditions, human brain membrane raft proteins were successfully resolved by RP-HPLC and some of the protein components, including specific integral membrane proteins, identified by downstream SDS-PAGE combined with in-gel digestion, or in-solution digestion and LC-MS/MS analysis of tryptic fragments. Using the described method, total protein recovery was high, and the repeatability of the separation maintained after repeated injections of membrane raft preparations.     

Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.