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.     

Selecting protein N-terminal peptides by combined fractional diagonal chromatography

In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines--which can include stable isotope labeling--occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography-tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ~5 d.     

Phosphopeptides enrichment using on-line two-dimensional strong cation exchange followed by reversed-phase liquid chromatography/mass spectrometry

We have developed a method to isolate and enhance the detection of phosphopeptides using liquid chromatography (LC)/mass spectrometry on a tryptic-digested protein sample. The method uses an on-line two-dimensional chromatography approach that consists of strong cation exchange (SCX) followed by reversed-phase (RP) chromatography with mass spectrometric detection. At pH 2.6 or lower, tryptic phosphopeptides are not retained during the first-dimension SCX chromatography step. Thus the capture of these peptides in the flow-through by the second-dimension RP trap can dramatically reduce the complexity of the phosphopeptide chromatography, resulting in little or no suppression of the signal often caused by the coeluting nonphosphorylated peptides. The method provides higher phosphopeptide recovery and less nonspecific biding of acidic peptides than the commonly used enrichment methods, such as immobilized metal affinity chromatography. Since the widely adopted multidimensional LC strategy in shotgun proteomics uses a similar SCX-RP approach, the method can be adapted to detect and characterize phosphopeptides from a complex mixture in a single experiment. Limitations of the method are also discussed.     

Integrated SDS removal and peptide separation by strong-cation exchange liquid chromatography for SDS-assisted shotgun proteome analysis

We report an improved shotgun method for analyzing proteomic samples containing sodium dodecyl sulfate (SDS). This method is based on the use of strong-cation exchange (SCX) liquid chromatography (LC) for SDS removal that can be integrated with peptide separation as the first dimension of the two-dimensional LC tandem mass spectrometry workflow. To optimize the performance of SDS removal, various experimental conditions, including the concentrations of chemical reagents and salts in the sample, the SDS concentration, and the SCX mobile phase composition, were investigated. It was found that a peptide recovery rate of about 90% could be achieved while removing SDS efficiently. One key finding was that, by increasing the SDS concentration to a certain level (0.5%) in the digested peptide sample, the sample recovery rate could be increased. The peptide recovery rate of BSA digests was found to be 90.6 ± 1.0% (n = 3), and SDS in the SCX fractions collected was not detectable by pyrolysis GC-MS, i.e., below the detection limit of 0.00006% for the undesalted SCX fractions. The peptide recovery rates were found to be 90.9% ± 2.7 (n = 3) and 89.5% ± 0.5% (n = 3) for the digests of the membrane-protein-enriched fractions of E. coli cell lysates and the MCF-7 breast cancer cell line, respectively. Compared to the methods that use acid-labile surfactants, such as RapiGest and PPS, for the MCF-7 membrane fraction sample, the SDS method identified, on average (n = 3), more peptides (～5%) and proteins (～16%) than the RapiGest method, while the RapiGest method identified more peptides (～21%) and proteins (～7%) from the E. coli membrane fraction than the SDS method. In both cases, the two methods identified more peptides and proteins than the PPS method. Since SCX is widely used as the first dimension of 2D-LC MS/MS, integration of SDS removal with peptide separation in SCX does not add any extra steps to the sample handling process. We demonstrated the application of this method for 2D-LC MS/MS profiling of the MCF-7 membrane protein fraction and identified 6889 unique peptides, corresponding to 2258 unique proteins or protein groups from two replicate experiments with a false peptide discovery rate of ～0.8%, compared to 5172 unique peptides and 1847 unique proteins identified by the RapiGest method.     

Comparison of ERLIC-TiO2, HILIC-TiO2, and SCX-TiO2 for global phosphoproteomics approaches

Reversible phosphorylations play a critical role in most biological pathways. Hence, in signaling studies great effort has been put into identification of a maximum number of phosphosites per experiment. Mass spectrometry (MS)-based phosphoproteomics approaches have been proven to be an ideal analytical method for mapping of phosphosites. However, because of sample complexity, fractionation of phosphopeptides prior to MS analysis is a crucial step. In the current study, we compare the chromatographic strategies electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), hydrophilic interaction liquid chromatography (HILIC), and strong cation exchange chromatography (SCX) for their fractionation behavior of phosphopeptides. In addition, we investigate the use of repetitive TiO(2)-based enrichment steps for a maximum identification of phosphopeptides. On the basis of our results, SCX yields the highest number of identified phosphopeptides, whereas ERLIC is optimal for the identification of multiphosphorylated peptides. Consecutive incubations of fractions and flow-through by TiO(2) beads enrich qualitatively different sets of phosphopeptides, increasing the number of identified phosphopeptides per analysis.     

A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics

Recently, we have developed a high-resolution two-dimensional separation strategy for the analysis of complex peptide mixtures. This methodology employs isoelectric focusing of peptides on immobilized pH gradient (IPG) gels in the first dimension, followed by reversed-phase chromatography in the second dimension, and subsequent tandem mass spectrometry analysis. The traditional approach to this mixture problem employs strong-cation-exchange (SCX) chromatography in the first dimension. Here, we present a direct comparison of these two first-dimensional techniques using complex protein samples derived from the testis of Rattus norvegicus. It was found that the use of immobilized pH gradients (narrow range pH 3.5-4.5) for peptide separation in the first dimension yielded 13% more protein identifications than the optimized off-line SCX approach (employing the entire pI range of the sample). In addition, the IPG technique allows for a much more efficient use on mass spectrometer analysis time. Separation of a tryptic digest derived from a rat testis sample on a narrow range pH gradient (over the 3.5-4.5 pH range) yielded 7626 and 2750 peptides and proteins, respectively. Peptide and protein identification was performed with high confidence using SEQUEST in combination with a data filtering program employing pI and statistical based functions to remove false-positives from the data.     

Perfluorooctanoic acid for shotgun proteomics

Here, we describe the novel use of a volatile surfactant, perfluorooctanoic acid (PFOA), for shotgun proteomics. PFOA was found to solubilize membrane proteins as effectively as sodium dodecyl sulfate (SDS). PFOA concentrations up to 0.5% (w/v) did not significantly inhibit trypsin activity. The unique features of PFOA allowed us to develop a single-tube shotgun proteomics method that used all volatile chemicals that could easily be removed by evaporation prior to mass spectrometry analysis. The experimental procedures involved: 1) extraction of proteins in 2% PFOA; 2) reduction of cystine residues with triethyl phosphine and their S-alkylation with iodoethanol; 3) trypsin digestion of proteins in 0.5% PFOA; 4) removal of PFOA by evaporation; and 5) LC-MS/MS analysis of the resulting peptides. The general applicability of the method was demonstrated with the membrane preparation of photoreceptor outer segments. We identified 75 proteins from 1 μg of the tryptic peptides in a single, 1-hour, LC-MS/MS run. About 67% of the proteins identified were classified as membrane proteins. We also demonstrate that a proteolytic (18)O labeling procedure can be incorporated after the PFOA removal step for quantitative proteomic experiments. The present method does not require sample clean-up devices such as solid-phase extractions and membrane filters, so no proteins/peptides are lost in any experimental steps. Thus, this single-tube shotgun proteomics method overcomes the major drawbacks of surfactant use in proteomic experiments.     

Evaluation and optimization of ZIC-HILIC-RP as an alternative MudPIT strategy

In proteomics, a digested cell lysate is often too complex for direct comprehensive mass spectrometric analysis. To reduce complexity, several peptide separation techniques have been introduced including very successful two-dimensional liquid chromatography (2D-LC) approaches. Here, we assess the potential of zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) as a first dimension for the analysis of complex peptide mixtures. We show that ZIC-HILIC separation is dramatically dependent on buffer pH in the range from 3 to 8, due to deprotonation of acidic amino acids. ZIC-HILIC exhibits a mixed-mode effect consisting of electrostatic and polar interactions. We developed a 2D-LC system that hyphenates ZIC-HILIC off-line with reversed-phase (RP). The two dimensions are fairly orthogonal, and the system performs very well in the analysis of minute amounts of complex peptide mixtures. Applying this method to the analysis of 10 mug of a cellular nuclear lysate, we were able to confidently identify over 1000 proteins. Compared to strong cation exchange chromatography (SCX), ZIC-HILIC shows better chromatographic resolution and absence of clustering of prevalent +2 and +3 charged peptides. At pH 3, ZIC-HILIC separation allows best orthogonality with RP and resembles conventional SCX separation. A significant enrichment of N-acetylated peptides in the first fractions is observed at these conditions. ZIC-HILIC separation at high pH (6.8 and 8), however, enables better chromatography, resulting in more comprehensive data acquisition. With this extended flexibility, we conclude that ZIC-HILIC is a very good alternative for the more conventional SCX in multidimensional peptide separation strategies.     

Comparative analysis of OFFGel,strong cation exchange with pH gradient,and RP at high pH for first‐dimensional separation of peptides from a membrane‐enriched protein fraction

The analysis and quantitation of membrane proteins have proved challenging for proteomics. Although several approaches have been introduced to complement gel‐based analysis of intact proteins, the literature is rather limited in comparing major emerging approaches. Peptide fractionation using IEF (OFFGel), strong cation exchange HPLC using a pH gradient (SCX‐pG), and RP HPLC at high pH, have been shown to increase peptide and protein identification over classic MudPIT approaches. This article compares these three approaches for first‐dimensional separation of peptides using a detergent phase (Triton X‐114) enriched membrane fraction from mouse cortical brain tissue. Results indicate that RP at high pH (pH 10) was superior for the identification of more peptides and proteins in comparison to the OFFGel or the SCX‐pG approaches. In addition, gene ontology analysis (GOMiner) revealed that RP at high pH (pH 10) successfully identified an increased number of proteins with “membrane” ontology, further confirming its suitability for membrane protein analysis, in comparison to SCX‐pG and OFFGel techniques.     

Strong cation exchange (SCX) based analytical methods for the targeted analysis of protein post-translational modifications

The multidimensional combination of strong cation exchange (SCX) chromatography and reversed phase chromatography has emerged as a powerful approach to separate peptides originating from complex samples such as digested cellular lysates or tissues before analysis by mass spectrometry, enabling the identification of over 10,000s of peptides and thousands of proteins in a single sample. Although, such multidimensional chromatography approaches are powerful, the in-depth analysis of protein post-translational modifications still requires additional sample preparation steps, involving the specific enrichment of peptides displaying the targeted modification. Here, we describe how in particular SCX chromatography can be used for the targeted analysis of important post-translational modifications, such as phosphorylation and N-terminal acetylation. Compared to other methods, SCX is less labor-intensive and more robust, and therefore likely more easily adaptable to main-stream research laboratories.     

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.     

Structural analysis of a prokaryotic ribosome using a novel amidinating cross-linker and mass spectrometry

The structure of the Escherichia coli ribosome, a 2.5 MDa ribonucleoprotein complex containing more than 50 proteins, was probed using the novel amidinating cross-linker diethyl suberthioimidate (DEST) and mass spectrometry. Peptide cross-links derived from this complex structure were identified at high confidence (FDR 0.8%) from precursor mass measurements and collision-induced dissociation (CID) fragmentation spectra. The acquired cross-linking data were found to be in excellent agreement with the crystal structure of the E. coli ribosome. DEST cross-links are particularly amenable to strong cation exchange (SCX) chromatography, facilitating a large-scale analysis. SCX enrichment and fractionation were shown to increase the number of cross-link spectra matches in our analysis 10-fold. Evidence is presented that these techniques can be used to study complex interactomes.     

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.     

Investigation of an on-line two-dimensional chromatographic approach for peptide analysis in plasma by LC–MS–MS

Reversed phase and hydrophilic interaction chromatography (HILIC) were successfully coupled for the on-line extraction and quantitative analysis of peptides by ESI–LC–MS/MS. A total of 11 peptides were utilized to determine the conditions for proper focusing and separation on both dimensions. Minor modifications to the initial organic composition of the first reversed-phase dimension provided options between a comprehensive (generic) or more selective approach for peptide transfer to the second HILIC dimension. Ion-pairing with trifluoroacetic acid (TFA) provided adequate chromatographic retention and peak symmetry for the selected peptides on both C₁₈ and HILIC. The resulting signal suppression from TFA was partially recovered by a post-column “TFA fix” using acetic acid yielding improvements in sensitivity. Minimal sample preparation aligned with standard on-line extraction procedures provided highly reproducible and robust results for over 300 sequential matrix injections. Final optimized conditions were successfully employed for the quantitation of peptide PTHrP (1–36) in rat K₃EDTA plasma from 25.0 to 10,000 ng/mL using PTHrP (1–34) as the analog internal standard. This highly orthogonal two-dimensional configuration was found to provide the unique selectivity required to overcome issues with interfering endogenous components and minimize electrospray ionization effects in biological samples.     

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.     

Combinatorial use of offline SCX and online RP-RP liquid chromatography for iTRAQ-based quantitative proteomics applications

Extensive front-end separation is usually required for complex samples in bottom-up proteomics to alleviate the problem of peptide undersampling. Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based experiments have particularly higher demands, in terms of the number of duty cycles and the sensitivity, to confidently quantify protein abundance. Strong cation exchange (SCX)/reverse phase (RP) liquid chromatography (LC) is currently used routinely to separate iTRAQ-labeled peptides because of its ability to simultaneously clean up the iTRAQ reagents and byproducts and provide first-dimension separation; nevertheless, the low resolution of SCX means that peptides can be redundantly sampled across fractions, leading to loss of usable duty cycles. In this study, we explored the combinatorial application of offline SCX fractionation with online RP-RP applied to iTRAQ-labeled chloroplast proteins to evaluate the effect of three-dimensional LC separation on the overall performance of the quantitative proteomics experiment. We found that the higher resolution of RP-RP can be harnessed to complement SCX-RP and increase the quality of protein identification and quantification, without significantly impacting instrument time and reproducibility.     

Novel strategy of high-abundance protein depletion using multidimensional liquid chromatography

In this study, for the first time, a comprehensive two-dimensional (2D) liquid-phase separation system, coupling strong cation exchange chromatography (SCX) to reversed-phase high performance liquid chromatography (RPLC), instead of specificity depletion method, was developed at the intact protein level for depletion of high-abundance proteins from rat liver. Proteins were prefractionated by SCX in the first dimensional separation, followed by RPLC with high resolution separation. UV absorption intensity was used to differentiate high-abundance proteins. The proteins with the absorbance intensity above 0.1 AU were defined as high abundance proteins and depleted. After removal of high-abundance proteins; other proteins were pooled, digested, and subsequently separated by capillary liquid chromatography coupled with MALDI-TOF/TOF mass spectrometry analysis. The high efficiency of the strategy was demonstrated by analyzing the soluble protein extracted from rat liver tissue. In total, 77 high-abundance proteins were depleted in one experiment flow. The ratio of depleted content of high-abundance proteins to that of total proteins was about 34.5%. In total, 1530 proteins were identified using the depletion strategy. Quantitative estimation of high-abundance proteins through liquid chromatography combined with UV absorption spectra was achieved. On the basis of the reproducible experimental results, a rapid and high-throughput depletion protocol was put forward. Along with depletion of the most (79.1%) high-abundance proteins and the separation of digested peptides, the total separation time could be less than 30 h. This strategy has no bias for depleting high-abundance proteins and enhances the number of identified proteins; therefore, it can be widely used in the global proteins analysis.     

Comparison of the sensitivity of evaporative universal detectors and LC/MS in the HILIC and the reversed-phase HPLC modes

It was hypothesized that the hydrophilic interaction liquid interface chromatography (HILIC) mode should produce more response than the reversed-phase HPLC mode on detectors with an evaporative component to the detection process. HILIC mobile phases are mostly composed of polar organic solvent and are more volatile than reversed-phase mobile phases. Therefore the more easily evaporated HILIC mobile phases should produce greater sensitivity for those detectors that remove mobile phase by evaporation. The responses of 12 compounds were measured in the reversed-phase mode and the HILIC mode with three detectors: evaporative light scattering detector (ELSD), corona charged aerosol detector (cCAD), and electrospray mass spectrometry (ESI-MS). The compounds studied were very polar compounds that were retained in the HILIC mode. Generally, the HILIC mode was able to achieve greater sensitivity than the reversed-phase mode for these compounds. The increases in sensitivity observed can be attributed to the more volatile HILIC mobile phase. For the ELSD, the HILIC mode produced slightly greater sensitivity than the reversed-phase mode. The cCAD was approximately 10 times more sensitive in the HILIC mode and the ESI-MS was approximately 5–10 times more sensitive in the HILIC mode. There was one instance in the study where a compound produced more response in the reversed-phase mode. Thymine yielded more sensitivity in the reversed-phase mode with the ESI-MS detector. In a given mode of operation, there was significant variation in the measured response factors for all compounds on each detector. While this is not unexpected for the ESI-MS detector, variation in the response factors between compounds indicates that the cCAD and ELSD are not truly universal detectors in the sense that all compounds have identical responses.     

Protein expression profiling of CLL B cells using replicate off-line strong cation exchange chromatography and LC-MS/MS

In this study we use replicate 2D-LC-MS/MS analyses of crude membranes from B cells derived from a patient with chronic lymphocytic leukemia (CLL) to examine the protein expression profile of CLL B cells. Protein identifications made by replicate 2D-LC-MS/MS analysis of tryptic peptides from detergent solubilized B cell membrane proteins, as well as replicate LC-MS/MS analysis of single off-line strong cation exchange chromatography (SCX) fractions, were analyzed. We show that despite the variance in SCX, capillary LC, and the data-dependent selection of precursor ions, an overlap of 64% between proteins identified in replicate runs was achieved for this system.     

Proteomic analysis of human ventricular cerebrospinal fluid from neurologically normal, elderly subjects using two-dimensional LC-MS/MS

A two-dimensional liquid chromatography separation scheme coupled to tandem mass spectrometry (2-D LC-MS/MS) was utilized to profile the proteome of human CSF. Ventricular CSF samples acquired post-mortem from 10 cognitively normal elderly subjects (mean +/- SEM Braak stage = 1.7 +/- 0.2) were analyzed to determine their protein composition. Raw CSF samples were subjected to an immunobased processing method to remove highly abundant albumin and immunoglobulin (Ig), allowing better detection of lower-abundance proteins. Samples were subjected to trypsin proteolysis followed by C18 solid-phase extraction. Tryptic CSF peptides were separated using a 2-D LC column, in which both strong cation exchange (SCX) and C18 phases were packed into a single capillary. MS/MS spectra of CSF peptides were searched against a human sub-database of the NBCI nonredundant database using the SEQUEST algorithm. Search results were further filtered using DTAselect, and individual samples were compared to one another using Contrast. Using this method, we were able to unambiguously identify 249 CSF proteins from 10 subjects. Of these proteins, 38% were unique to individual subjects, whereas only 6% were common to all 10 subjects. These results suggest considerable subject-to-subject variability in the CSF proteome.