Combinatorial Use of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) and Strong Cation Exchange (SCX) Chromatography for In-Depth Phosphoproteome Analysis

In large-scale phosphoproteomics studies, fractionation by strong cation exchange (SCX) or electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) is commonly used to reduce sample complexity, fractionate phosphopeptides from their unmodified counterparts, and increase the dynamic range for phosphopeptide identification. However, these procedures do not succeed to separate, both singly and multiply phosphorylated peptides due to their inverse physicochemical characteristics. Hence, depending on the chosen method only one of the two peptide classes can be efficiently separated. Here, we present a novel strategy based on the combinatorial separation of singly and multiply phosphorylated peptides by SCX and ERLIC for in-depth phosphoproteome analysis. In SCX, mostly singly phosphorylated peptides are retained and fractionated while not-retained multiply phosphorylated peptides are fractionated in a subsequent ERLIC approach (SCX-ERLIC). In ERLIC, multiply phosphorylated peptides are fractionated, while not-retained singly phosphorylated peptides are separated by SCX (ERLIC-SCX). Compared to single step fractionations by SCX, the combinatorial strategies, SCX-ERLIC and ERLIC-SCX, yield up to 48% more phosphopeptide identifications as well as a strong increase in the number of detected multiphosphorylated peptides. Phosphopeptides identified in two subsequent, complementary fractionations had little overlap (5%) indicating that ERLIC and SCX are orthogonal methods ideally suited for in-depth phosphoproteome studies.     

Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) versus strong cation exchange (SCX) for fractionation of iTRAQ-labeled peptides

The iTRAQ technique is popular for the comparative analysis of proteins in different complex samples. To increase the dynamic range and sensitivity of peptide identification in shotgun proteomics, SCX chromatography is generally used for the fractionation of iTRAQ-labeled peptides before LC-MS/MS analysis. However, SCX suffers from clustering of similarly charged peptides and the need to desalt fractions. In this report, SCX is compared with the alternative ERLIC method for fractionating iTRAQ-labeled peptides. The simultaneous effect of electrostatic repulsion and hydrophilic interaction in ERLIC results in peptide elution in order of decreasing pI and GRAVY values (increasing polarity). Volatile solvents can be used. We applied ERLIC to iTRAQ-labeled peptides from rat liver tissue, and 2745 proteins and 30,016 unique peptides were identified with high confidence from three technical replicates. This was 12.9 and 49.4% higher, respectively, than was obtained using SCX. In addition, ERLIC is appreciably better at the identification of highly hydrophobic peptides. The results indicate that ERLIC is a more convenient and more effective alternative to SCX for the fractionation of iTRAQ-labeled peptides. Quantification data show that both SCX and ERLIC fractionation have no significant effect on protein quantification by iTRAQ.     

Development and application of a phosphoproteomic method using electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), IMAC, and LC-MS/MS analysis to study Marek's Disease Virus infection

Marek's Disease (MD) is an avian neoplastic disease caused by Marek's Disease Virus (MDV). The mechanism of virus transition between the lytic and latent cycle is still being investigated; however, post-translational modifications, especially phosphorylation, have been thought to play an important role. Previously, our group has used strong cation exchange chromatography in conjunction with reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the changes in global proteomic expression upon MDV infection (Ramaroson , M. F.; Ruby, J.; Goshe, M. B.; Liu , H.-C. S. J. Proteome Res. 2008, 7, 4346-4358). Here, we extend our study by developing an effective separation and enrichment approach to investigate the changes occurring in the phosphoproteome using electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) to fractionate peptides from chicken embryo fibroblast (CEF) digests and incorporating a subsequent IMAC enrichment step to selectively target phosphorylated peptides for LC-MS/MS analysis. To monitor the multidimensional separation between mock- and MDV-infected CEF samples, a casein phosphopeptide mixture was used as an internal standard. With LC-MS/MS analysis alone, no CEF phosphopeptides were detected, while with ERLIC fractionation only 1.2% of all identified peptides were phosphorylated. However, the incorporation of IMAC enrichment with ERLIC fractionation provided a 50-fold increase in the percentage of identified phosphopeptides. Overall, a total of 581 unique phosphopeptides were identified (p < 0.05) with those of the MDV-infected CEF sample containing nearly twice as many as the mock-infected control of which 11% were unique to MDV proteins. The changes in the phosphoproteome are discussed including the role that microtubule-associated proteins may play in MDV infection mechanisms.     

Improved titanium dioxide enrichment of phosphopeptides from HeLa cells and high confident phosphopeptide identification by cross-validation of MS/MS and MS/MS/MS spectra

Enrichment is essential for phosphoproteome analysis because phosphorylated proteins are usually present in cells in low abundance. Recently, titanium dioxide (TiO2) has been demonstrated to enrich phosphopeptides from simple peptide mixtures with high specificity; however, the technology has not been optimized. In the present study, significant non-specific bindings were observed when proteome samples were applied to TiO2 columns. Column wash with an NH4Glu solution after loading peptide mixtures significantly increased the efficiency of TiO2 phosphopeptide enrichment with a recovery of up to 84%. Also, for proteome samples, more than a 2-fold increase in unique phosphopeptide identifications has been achieved. The use of NH4Glu for a TiO2 column wash does not significantly reduce the phosphopeptide recovery. A total of 858 phosphopeptides corresponding to 1034 distinct phosphosites has been identified from HeLa cells using the improved TiO2 enrichment procedure in combination with data-dependent neutral loss nano-RPLC-MS2-MS3 analysis. While 41 and 35% of the phosphopeptides were identified only by MS2 and MS3, respectively, 24% was identified by both MS2 and MS3. Cross-validation of the phosphopeptide assignment by MS2 and MS3 scans resulted in the highest confidence in identification (99.5%). Many phosphosites identified in this study appear to be novel, including sites from antigen Ki-67, nucleolar phosphoprotein p130, and Treacle protein. The study also indicates that evaluation of confidence levels for phosphopeptide identification via the reversed sequence database searching strategy might underestimate the false positive rate.     

A comparative study of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) versus SCX-IMAC-based methods for phosphopeptide isolation/enrichment

Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) has been introduced recently for phosphopeptide enrichment. Here we compared ERLIC with the well-established SCX-IMAC for identifying phosphopeptides in EGF-treated A431 cells. The ERLIC approach detected a higher number of phosphopeptides (17 311) than SCX-IMAC (4850), but it only detected 926 unique phosphopeptides compared to 1315 in SCX-IMAC. Only 12% unique phosphopeptides were common to both approaches, suggesting that more comprehensive phosphoproteomes could be generated by complementing SCX-IMAC with ERLIC.     

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.     

Simultaneous analysis of proteome, phospho- and glycoproteome of rat kidney tissue with electrostatic repulsion hydrophilic interaction chromatography

Protein post-translational modifications (PTMs) are regulated separately from protein expression levels. Thus, simultaneous characterization of the proteome and its PTMs is pivotal to an understanding of protein regulation, function and activity. However, concurrent analysis of the proteome and its PTMs by mass spectrometry is a challenging task because the peptides bearing PTMs are present in sub-stoichiometric amounts and their ionization is often suppressed by unmodified peptides of high abundance. We describe here a method for concurrent analysis of phosphopeptides, glycopeptides and unmodified peptides in a tryptic digest of rat kidney tissue with a sequence of ERLIC and RP-LC-MS/MS in a single experimental run, thereby avoiding inter-experimental variation. Optimization of loading solvents and elution gradients permitted ERLIC to be performed with totally volatile solvents. Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue. Two hundred low-abundance proteins with important functions were identified only from the glyco- or phospho-subproteomes, reflecting the importance of the enrichment and separation of modified peptides by ERLIC. In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein. Interestingly, partially modified proteins tend to occur on proteins involved in transport. Moreover, some membrane or extracellular proteins, such as versican core protein and fibronectin, were found to have both phosphorylation and N-glycosylation, which may permit an assessment of the potential for cross talk between these two vital PTMs and their roles in regulation.     

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

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

IMAC/TiO(2) enrich for peptide modifications other than phosphorylation: implications for chromatographic choice and database searching in phosphoproteomics

Protein regulation by reversible phosphorylation is fundamental in nature, and large-scale phosphoproteomic analyses are becoming routine in proteomics laboratories. These analyses utilise phosphopeptide separation and enrichment techniques linked to LC-MS/MS. Herein, we report that IMAC and TiO(2) also enrich for non-phosphorylated modified peptides such as acetylated, deamidated and carbamylated peptides. Urea and digestion conditions commonly used in phosphoproteomic workflows are the likely sources of the induced modifications (deamidation and carbamylation) and can easily modify phosphopeptides. Including these variable modifications in database searches increased the total number of identified phosphopeptides by 15%. We also show that strong cation exchange fractionation provides poor resolution of phosphopeptides and actually enriches these alternatively modified peptides. By switching to reverse-phase chromatography, we show a significant improvement in the number of identified phosphopeptides. We recommend that the users of phosphopeptide enrichment strategies avoid using urea as a denaturant and that careful consideration is given to chromatographic conditions and the types of variable modifications used in database searches. Thus, the capacity of IMAC and TiO(2) to enrich phosphopeptides bearing modifications other than phosphorylation is a previously unappreciated property of these chromatographies with practical implications for the field of phosphoproteomics.     

Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment

Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti(4+)-immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO(2) enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improve the enrichment of phosphopeptides containing multiple basic residues. We found that Ti(4+)-IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 μg of a HeLa cell lysate digest. In comparison, ～ 2000 unique phosphopeptides could be identified by Ti(4+)-IMAC with HFP and close to 3000 by TiO(2). We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti(4+)-IMAC methodology alongside the use of collision-induced dissociation (CID), higher energy collision induced dissociation (HCD) and electron transfer dissociation with supplementary activation (ETD) on considerably more complex sample, consisting of a total of 400 μg of triple dimethyl labeled MCF-7 digest. This analysis led to the identification of over 9,000 unique phosphorylation sites. The use of three peptide activation methods confirmed that ETD is best capable of sequencing multiply charged peptides. Collectively, our data show that the combination of SCX and Ti(4+)-IMAC is particularly advantageous for phosphopeptides with multiple basic residues.     

Multidimensional strategy for sensitive phosphoproteomics incorporating protein prefractionation combined with SIMAC, HILIC, and TiO(2) chromatography applied to proximal EGF signaling

Comprehensive enrichment and fractionation is essential to obtain a broad coverage of the phosphoproteome. This inevitably leads to sample loss, and thus, phosphoproteomics studies are usually only performed on highly abundant samples. Here, we present a comprehensive phosphoproteomics strategy applied to 400 μg of protein from EGF-stimulated HeLa cells. The proteins are separated into membrane and cytoplasmic fractions using sodium carbonate combined with ultracentrifugation. The phosphopeptides were separated into monophosphorylated and multiphosphorylated pools using sequential elution from IMAC (SIMAC) followed by hydrophilic interaction liquid chromatography of the mono- and nonphosphorylated peptides and subsequent titanium dioxide chromatography of the HILIC fractions. This strategy facilitated the identification of >4700 unique phosphopeptides, while 636 phosphosites were changing following short-term EGF stimulation, many of which were not previously known to be involved in EGFR signaling. We further compared three different data processing programs and found large differences in their peptide identification rates due to different implementations of recalibration and filtering. Manually validating a subset of low-scoring peptides exclusively identified using the MaxQuant software revealed a large percentage of false positive identifications. This indicates that, despite having highly accurate precursor mass determination, peptides with low fragment ion scores should not automatically be reported in phosphoproteomics studies.     

Comprehensive identification of phosphorylation sites in postsynaptic density preparations

In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC. Initially, tryptic peptides were separated by cation exchange and analyzed by reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions. Because each of these individual fractions was too complex to characterize completely in single LC-MS/MS runs, we enriched for phosphopeptides by performing IMAC on each SCX fraction, yielding at least a 3-fold increase in identified phosphopeptides relative to either approach alone (SCX or IMAC). This enabled us to identify at least one site of phosphorylation on 23% (287 of 1,264) of all proteins found to be present in the postsynaptic density preparation. In total, we identified 998 unique phosphorylated peptides, mapping to 723 unique sites of phosphorylation. At least one exact site of phosphorylation was determined on 62% (621 of 998) of all phosphopeptides, and approximately 80% of identified phosphorylation sites are novel.     

Global profiling of phosphopeptides by titania affinity enrichment

Protein phosphorylation is a ubiquitous post-translational modification critical to many cellular processes. Large-scale unbiased characterization of phosphorylation status remains a major technical challenge in proteomics. In the present work, we evaluate and optimize titania-based affinity enrichment for global profiling of phosphopeptides from complex biological mixtures. We demonstrate that inclusion of glutamic acid in the sample loading buffer substantially reduced nonspecific binding of nonphosphorylated peptides to the titania while retaining the high binding affinity for phosphopeptides. The reduction in nonspecific peptide binding enhanced overall phosphopeptide recovery, ranging from 22 to 85%, and led to substantial improvement in large-scale global profiling. In addition, we observed that the overall identification of phosphopeptides was significantly enhanced by neutral loss-triggered MS (3) scans and respective use of multiple charge- and mass-dependent filtering criteria for MS (2) and MS (3) spectra. In conjunction with strong-cation exchange chromatography (SCX) for prefractionation, a total of 4002 distinct phosphopeptides were identified from SKBr3 breast cancer cells at false-positive rates of 3.7% and 5.5%, respectively, for singly and doubly phosphorylated peptides.     

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.     

Antibody characterization using novel ERLIC-MS/MS-based peptide mapping

Electrostatic repulsion hydrophilic interaction chromatography (ERLIC) coupled with mass spectrometry (MS) is a technique that is increasingly being used as a trapping/enrichment tool for glycopeptides/phosphorylated peptides or sample fractionation in proteomics research. Here, we describe a novel ERLIC-MS/MS-based peptide mapping method that was successfully used for the characterization of denosumab, in particular the analysis of sequence coverage, terminal peptides, methionine oxidation, asparagine deamidation and glycopeptides. Compared to reversed phase liquid chromatography (RPLC)-MS/MS methods, ERLIC demonstrated unique advantages in the retention of small peptides, resulting in 100% sequence coverage for both the light and heavy chains. It also demonstrated superior performance in the separation and characterization of asparagine deamidated peptides, which is known to be challenging by RPLC-MS/MS. The developed method can be used alone for peptide mapping-based characterization of monoclonal antibodies, or as an orthogonal method to complement the RPLC-MS/MS method. This study extends the applications of ERLIC from that of a trapping/fractioning column to biologic therapeutics characterization. The ERLIC-MS/MS method can enhance biologic therapeutics analysis with more reliability and confidence for bottom-up peptide mapping-based characterization.     

TiSH - a robust and sensitive global phosphoproteomics strategy employing a combination of TiO(2), SIMAC, and HILIC

Large scale quantitative phosphoproteomics depends upon multidimensional strategies for peptide fractionation, phosphopeptide enrichment, and mass spectrometric analysis. Previously, most robust comprehensive large-scale phosphoproteomics strategies have relied on milligram amounts of protein. We have set up a multi-dimensional phosphoproteomics strategy combining a number of well-established enrichment and fraction methods: An initial TiO(2) phosphopeptide pre-enrichment step is followed by post-fractionation using sequential elution from IMAC (SIMAC) to separate multi- and mono-phosphorylated peptides, and hydrophilic interaction liquid chromatography (HILIC) of the mono-phosphorylated peptides (collectively abbreviated "TiSH"). The advantages of the strategy include a high specificity and sample preparation workload reduction due to the TiO(2) pre-enrichment step, as well as low adsorptive losses. We demonstrate the capability of this strategy by quantitative investigation of early interferon-γ signaling in low quantities of insulinoma cells. We identified ~6600 unique phosphopeptides from 300μg of peptides/condition (22 unique phosphopeptides/μg) in a duplex dimethyl labeling experiment, with an enrichment specificity>94%. When doing network analysis of putative phosphorylation changes it could be noted that the identified protein interaction network centered upon proteins known to be affected by the interferon-γ pathway, thereby supporting the utility of this global phosphoproteomics strategy. This strategy thus shows great potential for interrogating signaling networks from low amounts of sample with high sensitivity and specificity.     

Global analysis of protein phosphorylation networks in insulin signaling by sequential enrichment of phosphoproteins and phosphopeptides

Although the important role of protein phosphorylation in insulin signaling networks is well recognized, its analysis in vivo has not been pursued in a systematic fashion through proteome-wide studies. Here we undertake a global analysis of insulin-induced changes in the rat liver cytoplasmic and endosomal phosphoproteome by sequential enrichment of phosphoproteins and phosphopeptides. After subcellular fractionation proteins were denatured and loaded onto iminodiacetic acid-modified Sepharose with immobilized Al3? ions (IMAC-Al resin). Retained phosphoproteins were eluted with 50 mM phosphate and proteolytically digested. The digest was then loaded onto an IMAC-Al resin and phosphopeptides were eluted with 50 mM phosphate, and resolved by 2-dimensional liquid chromatography, which combined offline weak anion exchange and online reverse phase separations. The peptides were identified by tandem mass spectrometry, which also detected the phosphorylation sites. Non-phosphorylated peptides found in the flow-through of the IMAC-Al columns were also analyzed providing complementary information for protein identification. In this study we enriched phosphopeptides to ~85% purity and identified 1456 phosphopeptides from 604 liver phosphoproteins. Eighty-nine phosphosites including 45 novel ones in 83 proteins involved in vesicular transport, metabolism, cell motility and structure, gene expression and various signaling pathways were changed in response to insulin treatment. Together these findings could provide potential new markers for evaluating insulin action and resistance in obesity and diabetes.     

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.     

Enhancement of the efficiency of phosphoproteomic identification by removing phosphates after phosphopeptide enrichment

Immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2) chromatography are simple, widely used, and cost-effective methods to enrich phosphopeptides, but the sample loading buffer composition, desalting procedure, and control of loading amount are critical to avoid nonspecific interactions and to achieve efficient phosphopeptide enrichment. Although the combination of MS3 analysis and high-resolution mass spectrometry (MS) is helpful to identify phosphopeptides, the quality of many MS/MS spectra having a neutral loss peak of phosphate is still too poor to allow sequence identification, and this results in many false-negative as well as false-positive identifications. Here, we present a novel strategy, which is based on the use of alkaline phosphatase to remove phosphates and analysis of phospho/dephosphopeptide retention times to increase the reliability of identification. The use of phospho/dephosphopeptide retention time ratios allows the identification of phosphopeptides with high confidence with the aid of a focused database of dephosphopeptides. This approach was very effective to identify multiple phophorylations in tryptic peptides. A 'true' phosphorylation data set should contain about 90% phospho-Ser and a few percent phospho-Tyr, and this ratio can be used as a quality criterion for evaluation of data sets. By applying this efficient approach, we were able to identify more than one thousand phosphopeptides.     

Shotgun proteomics of the haloarchaeon Haloferax volcanii

Haloferax volcanii, an extreme halophile originally isolated from the Dead Sea, is used worldwide as a model organism for furthering our understanding of archaeal cell physiology. In this study, a combination of approaches was used to identify a total of 1296 proteins, representing 32% of the theoretical proteome of this haloarchaeon. This included separation of (phospho)proteins/peptides by 2-dimensional gel electrophoresis (2-D), immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and Multidimensional Protein Identification Technology (MudPIT) including strong cation exchange (SCX) chromatography coupled with reversed phase (RP) HPLC. Proteins were identified by tandem mass spectrometry (MS/MS) using nanoelectrospray ionization hybrid quadrupole time-of-flight (QSTAR XL Hybrid LC/MS/MS System) and quadrupole ion trap (Thermo LCQ Deca). Results indicate that a SCX RP HPLC fractionation coupled with MS/MS provides the best high-throughput workflow for overall protein identification.