Nonretentive solid-phase extraction of phosphorylated peptides from complex peptide mixtures for detection by matrix-assisted laser desorption/ionization mass spectrometry

Widespread interest in protein phosphorylation has led to the development of a variety of methods for the analysis of phosphoproteomes of different types of organisms. Many applications involve pretreatment of the sample before mass spectrometric measurement and can crucially improve the detection efficiency of individual phosphopeptides. Despite intense research efforts, separation and extraction of phosphorylated peptides, especially multiphosphorylated ones, remain challenging tasks and need to be further explored and expanded with unconventional approaches. In this study, we describe the application of nonretentive solid-phase extraction (SPE) to the analysis of phosphopeptides using the highly cross-linked polystyrene-divinylbenzene material Strata-X. This study indicates that the procedure allows for the preferential extraction of phosphopeptides regardless of their extent of phosphorylation. The Strata-X material primarily retains nonphosphorylated peptides by hydrophobic interaction, whereas the inherent hydrophilicity of phosphorylated peptides leads to their partitioning into the aqueous phase. Phosphopeptides that were rapidly segregated out of tryptic digest mixtures and collected in the early aqueous fractions generated intense signals in mass spectra. The method was developed using SPE Strata-X columns, then suited for detection and sequencing of phosphopeptides by miniaturizing the system to the scale of custom-made microcolumns. This provided fast isolation of phosphopeptides from protein digests along with direct MALDI on-target deposition. The possibility of on-target washing during sample preparation is also presented.     

Detection of multiphosphorylated peptides in LC-MS/MS analysis under low pH conditions

An improved method of detection of multiphosphorylated peptides by RPLC-MS/MS analysis under low pH conditions (pH approximately 1.7, 3% formic acid) is demonstrated for the model phosphoproteins, bovine alpha- and beta-casein. Changes in the pH conditions from normal (pH approximately 3.0, 0.1% formic acid) to low (pH approximately 1.7, 3% formic acid) significantly improved the detection limit of multiphosphorylated peptides carrying negative (-) solution charge states. In particular, bovine beta-casein tetraphosphorylated peptide, was detected with a loading amount of only 50 fmol of trypsin-digested bovine beta-casein under low pH conditions, which is 200 times lower than necessary to detect the peptide under normal pH conditions. In order to understand the low pH effect, various loading amounts of trypsin-digested bovine alpha- and beta-caseins were analyzed by RPLC-MS/MS analyses under two different pH conditions. The question of whether the low pH condition improves the detection of multiphosphorylated peptides by increasing ionization efficiencies could not be proven in this study because synthetic multiphosphorylated peptides could not be easily obtained by peptide synthesis. Interestingly, increased hydrophilicity resulting from multiple phosphorylation events is shown to negatively affect the peptide retention on reversed-phase column material. It was also demonstrated that the low pH condition could effectively enhance the retention of multiphosphorylated peptides on reversed-phase column material. The usefulness of low pH RPLC analysis was tested using an actual phosphopeptide-enriched sample prepared from mouse brain tissues. Previously, low pH solvents have been used in SCX fractionation and TiO2 enrichment processes to selectively enrich phosphopeptides during the phosphopeptide enrichment procedure, but the improved detection of multiphosphorylated peptides in RPLC-MS/MS analysis under low pH conditions has not been reported before (Ballif, B. A.; Villen, J.; Beausoleil, S. A.; Schwartz, D.; Gygi, S. P. Mol. Cell. Proteomics 2004, 3, 1093-1101. Villen, J.; Beausoleil, S. A.; Gerber, S. A.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 1488-1493. Schlosser, A.; Vanselow, J. T.; Kramer, A. Anal. Chem. 2005, 77, 5243-5250.).     

Efficient identification of phosphorylation by mass spectrometric phosphopeptide fingerprinting

We describe a rapid and efficient method for the identification of phosphopeptides, which we term mass spectrometric (MS) phosphopeptide fingerprinting. The method involves quantitative comparison of proteolytic peptides from native versus completely dephosphorylated proteins. Dephosphorylation of serine, threonine, and tyrosine residues is achieved by in-gel treatment of the separated proteins with hydrogen fluoride (HF). This chemical dephosphorylation results in enrichment of those unmodified peptides that correspond to previously phosphorylated peptides. Quantitative comparison of the signal-to-noise ratios of peaks in the treated versus untreated samples are used to identify phosphopeptides, which can be confirmed and further studied by tandem mass spectrometry (MS/MS). We have applied this method to identify eight known phosphorylation sites of Xenopus Aurora A kinase, as well as several novel sites in the Xenopus chromosome passenger complex (CPC).     

A method for the chemical generation of N-terminal peptide sequence tags for rapid protein identification

We describe a method for generating multiple small sequences from the N terminal of peptides in unseparated protein digests by stepwise thioacetylation and acid cleavage. The mass differences between a series of N-terminally degraded peptides give short sequences of defined length. Such short "sequence tags" together with the mass of the parent peptide can be used to identify the protein in a database. The sequence ladders are generated without the use of chain terminators or sample aliquoting and the degradation reagents are water soluble so that the chemistry can be carried out on peptides immobilized on C-18 reversed-phase supports without any peptide loss due to washing with organic solvents as occurs in Edman type sequencing. The entire procedure can be automated, and we describe a prototype device for the parallel analysis of multiple samples. We demonstrate the effectiveness of this chemical tagging method in a comparison with Edman sequencing, peptide mass fingerprinting, and MS/MS analysis of crude protein fractions obtained from an HPLC separation of the Escherichia coli ribosome complex which consists of 57 proteins. We show that chemical tagging is a viable first-pass high-throughput identification method to be used prior to an in depth MS/MS analysis.     

Selective extraction and characterization of a histidine-phosphorylated peptide using immobilized copper(II) ion affinity chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Phosphorylation is the predominant posttranslational modification involved in regulating enzymatic activity and mediating signal transduction in prokaryotic and eukaryotic cells. Selective enrichment of phosphorylated peptides prior to mass spectrometric analysis facilitates identification of phosphorylated proteins, determination of specific phosphorylated residues, and characterization of the conditions under which phosphorylation occurs. Such protocols have been established for peptides containing residues that form phosphoesters, such as serine and threonine, using immobilized metal-ion affinity chromatography. Despite the importance of histidine phosphorylation in two-component signal transduction pathways, similar protocols for peptides containing phosphorylated histidine (P-His) residues have proven elusive, due to the instability of these modifications and the propensity of unphosphorylated histidines to interact with immobilized metals ions. We describe a method for the selective extraction of a P-His-containing peptide using immobilized copper(II) ions and disposable metal-chelating pipet tips (ZipTipMC, Millipore). The method is contingent upon pH-dependent interactions between the phosphate group and immobilized copper(II) ions. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with postsource decay confirms the identity and phosphorylation state of the extracted peptide. Peptides containing unphosphorylated histidine residues or other phosphorylated amino acids are not retained, demonstrating the specificity of the method for P-His-containing peptides.     

Ligand-exchange detection of phosphorylated peptides using liquid chromatography electrospray mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is used to selectively detect analytes with a high affinity for metal ions. The detection method is based on the selective monitoring of a competing ligand at its specific m/z value that is released during the ligand-exchange reaction of a metal-ligand complex with analyte(s) eluting from a reversed-phase liquid chromatography column. The ligand-exchange reaction proceeds in a postcolumn reaction detection system placed prior to the inlet of the electrospray MS interface. The feasibility of metal affinity detection by ESI-MS is demonstrated using phosphorylated peptides and iron(III)methylcalcein blue as reactant, as a model system. Methylcalcein blue (MCB) released upon interaction with phosphorylated peptides is detected at m/z 278. The ligand-exchange detection is coupled to a C8 reversed-phase column to separate several nonphosphorylated enkephalins and the phosphorylated peptides pp60 c-src (P) and M2170. Detection limits of 2 microM were obtained for pp60 c-src (P) and M2170. The linearity of the detection method is tested in the range of 2-80 micromol/L phosphorylated compounds (r(2) = 0.9996), and a relative standard deviation of less than 8% (n = 3) for all MCB responses of the different concentrations of phosphorylated compounds was obtained. The presented method showed specificity for phosphorylated peptides and may prove a useful tool for studying other ligand-exchange reactions and metal-protein interactions.     

Global protein identification and quantification technology using two-dimensional liquid chromatography nanospray mass spectrometry

A simple and reliable method is described here for the identification and relative quantification of proteins in complex mixtures using two-dimensional liquid chromatography/tandem mass spectrometry. The method is based on the classical proteomic analysis where proteins are digested with trypsin and the resulting peptides are separated by multidimensional liquid chromatography. The separated peptides are analyzed by tandem mass spectrometry and identified via a database search algorithm such as SEQUEST. The peak areas (integrated ion counts over the peptide elution time) of all identified peptides are calculated, and the relative concentration of each protein is determined by comparing the peak areas of all peptides from that protein in one sample versus those from the other. Using this strategy, we compared the relative level of protein expression of A431 cells (an epidermal cell line) grown in the presence or absence of epidermal growth factor (EGF). Our results are consistent with the published observations of the transient effects of EGF. In addition, the difference in the concentrations of several phosphopeptides determined in our studies suggests the possibility of several new targets involved in the EGF cell-signaling pathway. This global protein identification and quantification technology should prove to be a valuable means for comparing proteomes in biological samples subjected to differential treatments.     

Enhanced ionization of phosphorylated peptides during MALDI TOF mass spectrometry

Although alpha-cyano-4-hydroxycinnamic acid functions as an excellent matrix for the analysis of most peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry, the ionization of phosphorylated peptides is usually suppressed by nonphosphorylated peptides. As an alternative matrix, 2',4',6'-trihydroxyacetophenone (THAP) with diammonium citrate was found to overcome this problem for the MALDI TOF mass spectrometric analysis of proteolytic digests of phosphorylated proteins. Specifically, the abundances of phosphorylated peptides in tryptic digests of bovine beta-casein and protein kinase C (PKC)-treated mouse cardiac troponin I were enhanced more than 10-fold using THAP during positive ion MALDI TOF mass spectrometry. The protonated molecules of phosphorylated peptides were sufficiently abundant that postsource decay TOF mass spectrometry was used to confirm the number of phosphate groups in each peptide. Finally, tryptic digestion followed by analysis using MALDI TOF mass spectrometry with THAP as the matrix facilitated the identification of a unique phosphorylation site in PKC-treated troponin I.     

Identification and relative quantitation of protein mixtures by enzymatic digestion followed by capillary reversed-phase liquid chromatography-tandem mass spectrometry

In this report, we describe an approach for identification and relative quantitation of individual proteins within mixtures using LC/MS/MS analysis of protein digests. First, the proteins are automatically identified by correlating the tandem mass spectra with peptide sequences from a database. Then, peak areas of identified peptides from one protein are added together to define the total reconstructed peak area of the protein digest. The total reconstructed peak area is further normalized to the peak area of an internal standard protein digest present in the mixture at a constant level. The method was illustrated using digested mixtures of five standard proteins as follows. One protein was gradually diluted while the other four components were present in the mixtures at constant level. This study revealed that relative peak area of the variable protein increased linearly (trend line R2 = 0.9978) with increasing amount from 10 to 1000 fmol, while relative peak areas of four constant proteins remained approximately the same (within 20% relative standard deviation). To further evaluate the applicability of this method for the quantitation of proteins from complex mixtures, human plasma protein digest was spiked with 200 and 400 fmol of myoglobin digest. Total peak area of myoglobin peptides was normalized to the total peak area of apolipoprotein A-I peptides from human plasma, which played the role of an internal standard. The myoglobin/apolipoprotein A-I peak area ratio was 2 times larger for the human plasma digest spiked with a double amount of myoglobin. After several repetitions, the error of the relative peak area measurements remained below 11%, suggesting that the method described here can be used for relative concentration measurements of proteins in the complex biological mixtures. In the presented method, chemical derivatization steps are not needed to create an internal standard, as in isotope-coded affinity tag or similar methods.     

Detection of in vitro kinase generated protein phosphorylation sites using gamma[18O4]-ATP and mass spectrometry

A novel stable-isotope labeling approach for identification of phosphopeptides that utilizes adenosine triphosphate, in which four oxygen-16 atoms attached to the terminal phosphate group are substituted with oxygen-18 [gamma((18)O4)-ATP], has been developed. The ability to use gamma((18)O4)-ATP to monitor phosphorylation modification within various proteins was conducted by performing in vitro kinase reactions in the presence of a 1:1 mixture of gamma((18)O4)-ATP and normal isotopic abundance ATP (ATP). After tryptic digestion, the peptides were analyzed using mass spectrometry (MS). Phosphorylated peptides are easily recognized within the MS spectrum owing to the presence of doublets separated by 6.01 Da; representing versions of the peptide modified by ATP and gamma((18)O4)-ATP. Standard peptides phosphorylated using gamma((18)O4)-ATP via in vitro kinase reactions showed no exchange loss of (18)O with (16)O. The identity of these doublets as phosphorylated peptides could be readily confirmed using tandem MS. The method described here provides the first direct stable-isotope labeling method to definitely detect phosphorylation sites within proteins.     

Utility of cleavable isotope-coded affinity-tagged reagents for quantification of low-copy proteins induced by methylprednisolone using liquid chromatography/tandem mass spectrometry

Gene expression changes underlie important biological and pharmacological responses. Although mRNA expression profiling is routine, quantification of low-abundance proteins, which typically represent key effectors of responses, remains challenging. A novel strategy was developed for sensitive and accurate quantification of low-abundance proteins in highly complex biological matrixes. First, the cysteine specificity of cleavable isotope-coded affinity tags (cICAT) was employed to reduce the complexity of the digested proteome of tissue homogenates and to improve the quantification of low-abundance proteins. Second, cICAT-treated tissue samples were analyzed on a capillary LC coupled to an ion trap MS to screen for the subset of cICAT-peptides, derived from target proteins of interest, that was successfully labeled and retrieved. Third, putatively identified peptides derived from target proteins were synthesized, cICAT-labeled, and used both to optimize multiple reactions monitoring (MRM) analysis and to confirm chromatographic retention time and fragmentation pattern. Finally, batch quantification of target peptides was performed using MRM on a LC/triple-quad MS/MS using (12)C- (control) and (13)C (experimental)-cICAT-labeled tissue mixtures. The utility of this method was demonstrated by elucidating the time-course of tyrosine aminotransferase induction in the liver of rats following treatment with the corticosteroid methylprednisolone (MPL). This approach significantly improved quantitative sensitivity, and the linear range was 10-fold greater than published previously. An additional advantage is that archived samples may be reinterrogated to investigate the regulation of additional targets that become of interest. Stored samples were sucessfully reinterrogated to monitor the induction of ornithine decarboxylase, which is also an MPL-induced protein. To our knowledge, this is the first report of an ICAT-based method that is capable of quantifying low-abundance proteins in highly complex samples, such as tissue homogenates. The approach enables simultaneous quantification of multiple effector proteins induced by biological or pharmacological stimuli, and the processed samples can be interrogated repeatedly as additional targets of interest arise.     

Selective sampling of phosphopeptides for detection by MALDI mass spectrometry

The analysis of phosphopeptides by mass spectrometry (MS) is one of the most challenging tasks in proteomics. This is due to the lower isoelectric point (pI) of phosphopeptides, which leads to inefficient sample ionization in MS, particularly when competing with other peptides. The problem is compounded by the typical low abundance of phosphopeptides in biological samples. We describe here a simple nonsorptive method to isolate phosphopeptides based on their pI. A voltage is applied to selectively migrate the phosphopeptides into a capillary, which are negatively charged at acidic pH. The selectively sampled fraction is directly deposited onto MALDI sample target in nanoliter volumes (7-35 nL) for highly sensitive MS detection. No significant sample loss is evident in this procedure; hence, the MS was able to detect the isolated phosphopeptides at trace quantity. In this case, attomole-level detection limit is achieved for synthetic phosphopeptides (nM concentration and nL volume), from a mixture containing other peptides at up to 1 million times higher in concentration. Selective sampling was also applied to the tryptic digest of beta- and alpha-caseins to reveal the multiple phosphorylated peptides at the low-femtomole level using MALDI MS. Knowledge of pI based on the rejection/injection of peptides was found to be useful in peak assignment. To confirm the sequence of the selectively sampled peptides, fraction collection was performed for offline ESI MS/MS analysis.     

Quantification of intermediate-abundance proteins in serum by multiple reaction monitoring mass spectrometry in a single-quadrupole ion trap

A method is presented to quantify intermediate-abundance proteins in human serum using a single-quadrupole linear ion trap mass spectrometer-in contrast, for example, to a triple-quadrupole mass spectrometer. Stable-isotope-labeled (tryptic) peptides are spiked into digested protein samples as internal standards, aligned with the traditional isotope dilution approach. As a proof-of-concept experiment, four proteins of intermediate abundance were selected, coagulation factor V, adiponectin, C-reactive protein (CRP), and thyroxine binding globulin. Stable-isotope-labeled peptides were synthesized with one tryptic sequence from each of these proteins. The normal human serum concentration ranges of these proteins are from 1 to 30 microg/mL (or 20 to 650 pmol/mL). These labeled peptides and their endogenous counterparts were analyzed by LC-MS/MS using multiple reaction monitoring, a multiplexed form of the selected reaction monitoring technique. For these experiments, only one chromatographic dimension (on-line reversed-phase capillary column) was used. Improved limits of detection will result with multidimensional chromatographic methods utilizing more material per sample. Standard curves of the spiked calibrants were generated with concentrations ranging from 3 to 700 pmol/mL using both neat solutions and peptides spiked into the complex matrix of digested serum protein solution where ion suppression effects and interferences are common. Endogenous protein concentrations were determined by comparing MS/MS peak areas of the endogenous peptides to the isotopically labeled internal calibrants. The derived concentrations from a normal human serum pool (neglecting loss of material during sample processing) were 9.2, 110, 120, and 246 pmol/mL for coagulation factor V, adiponectin, CRP, and thyroxine binding globulin, respectively. These concentrations generally agree with the reported normal ranges for these proteins. As a measure of analytical reproducibility of this single-quadrupole assay, the coefficients of variance based on 12 repeated measurements for each of the endogenous tryptic peptides were 17.0, 25.4, 24.2, and 14.0% for coagulation factor V, adiponectin, CRP, and thyroxine binding globulin, respectively.     

Alpha-cyano-4-hydroxycinnamic acid affinity sample preparation. A protocol for MALDI-MS peptide analysis in proteomics

We present a new MALD1 sample preparation technique for peptide analysis using the matrix alpha-cyano-4-hydroxy-cinnamic acid (CHCA) and prestructured sample supports. The preparation integrates sample purification, based on the affinity of microcrystalline CHCA for peptides, thereby simplifying the analysis of crude peptide mixtures. Enzymatic digests can thus be prepared directly, without preceding purification. Prepared samples are homogeneous, facilitating automatic spectra acquisition. This method allows preparation of large numbers of samples with little effort and without the need for automation. These features make the described preparation suitable for cost-efficient high-throughput protein identification. Performance of the sample preparation is demonstrated with in situ proteolytic digests of human brain proteins separated by two-dimensional gel electrophoresis.     

Analysis of complex protein mixtures with improved sequence coverage using (CE-MS/MS)n

Identification of proteins, in a complex protein mixture, using one-dimensional high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analysis of its digest, usually suffers from low sequence coverage. There are several reasons for the low coverage including undersampling, wide concentration dynamic range of the proteins in a complex protein mixture, and wide range of electrospray ionization efficiency of peptides under each mobile-phase composition. To address this low sequence coverage, we introduce a novel technique, (CE-MS/MS)n, which utilizes the most significant advantages of CE-MS/MS, including economy of sample size, fast analysis time, and high separation efficiency, to increase the sequence coverage of a complex protein mixture. Based on these characteristics, (CE-MS/MS)n can be performed in which multiple CE-MS/MS subanalyses (injections followed by analyses) are analyzed and experimental variables are manipulated during each CE-MS/MS subanalysis in order to maximize sequence coverage. (CE-MS/MS)n is a practical technique since each CE-MS/MS subanalysis consumes <10 nL, and each CE-MS/MS subanalysis takes approximately 10 min; therefore, several subanalyses can be performed in approximately 1 h consuming only nanoliters of the sample. Two techniques have been introduced to address the undersampling: (1) (CE-MS/MS)n using dynamic exclusion. In this technique, several CE-MS/MS analyses (injection followed by separation) were performed in one run using the dynamic exclusion capability of the mass spectrometer until all peptide peaks were analyzed by MS/MS. (2) Gas-phase fractionation. In this technique, (CE-MS/MS)n is performed by scanning a narrow mass range (every approximately 100 m/z) during each CE-MS/MS subanalysis without using dynamic exclusion. Under this condition, in each subanalysis, the number of peptides available for MS/MS analysis is significantly reduced, and peptides with the same nominal masses are analyzed, thereby increasing sequence coverage. Additionally, to address the lack of detection of low-level peptides in a mixture containing a wide concentration dynamic range, the concentration of the sample was systematically increased in each subanalysis (while utilizing dynamic exclusion) so that low-intensity peptides would rise above the mass spectrometer threshold and, consequently, undergo MS/MS analysis. Moreover, to alter the ionization efficiency of peptides with low electrospray ionization efficiency, and to change the migration behavior of comigrating peptides under a specific liquid composition, the CE background electrolyte was modified in several subanalyses to further improve sequence coverage. The combination of the above-mentioned techniques was applied to the analysis of the tryptic digests of three well-characterized protein mixtures: a six-protein mixture with average MW of approximately 26,000 (standard I), a six-protein mixture with an average MW approximately 49,000 (standard II), and a more complex protein mixture containing 55 proteins (E. coli ribosomal proteins). In approximately 1 h, when the MS/MS of the peptides were manually checked, all peptides that produced peaks under electrospray ionization in the scanned range of the analysis (500-2000 m/z) and within the practical fragmentation capability of the MS (peptides with MW <3500) were identified for standard I by consuming only 200 fmol of each protein. When searched against a Swissprot database, the average sequence coverage for the standard I, II, and E. coli's ribosomal proteins were 57, 34, and 15%, respectively.     

Elution-modified displacement chromatography coupled with electrospray ionization-MS: on-line detection of trace peptides at low-femtomole level in peptide digests

Elution-modified displacement chromatography (EMDC) was employed to achieve peptide separations with high efficiency. On-line ESI-MS and ESI-MS/MS measurements showed enrichment and detection of kemptide, a protein kinase A peptide substrate, at low femtomole levels when it was added as a trace marker component to a tryptic digest of bovine serum proteins or to a human growth hormone peptide digest at concentration ratios between 1:10(5) and 1:10(6). In another EMDC separation, five peptides were detected in a mixture containing 20 fmol of human growth hormone tryptic digest mixed with the bovine serum protein digest. We found that EMDC facilitated rapid detection and sequence analysis of trace peptides at levels of approximately 0.5 fmol/microL in complex peptide mixtures with a wide dynamic concentration range. Accordingly, the detection of kemptide by EMDC was found to be 3-4 orders of magnitude more sensitive than that attained in conventional linear elution chromatography separations performed with the same peptide loads. Kemptide was phosphorylated in vitro and was detected along with its neutral loss product in peptide mixtures at low femtomole levels. EMDC enabled both detection and amino acid sequence determination on trace levels of phosphorylated and other posttranslationally modified peptides, suggesting that the technique may be useful for proteomics applications where detection and analysis of trace level peptides are problematic.     

Phosphoprotein isotope-coded solid-phase tag approach for enrichment and quantitative analysis of phosphopeptides from complex mixtures

Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated beta-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line.     

In situ liquid-liquid extraction as a sample preparation method for matrix-assisted laser desorption/ionization MS analysis of polypeptide mixtures

A novel liquid-liquid extraction (LLE) procedure was investigated for preparation of peptide and protein samples for matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). LLE using ethyl acetate as the water-immiscible organic solvent enabled segregation of hydrophobic and hydrophilic polypeptides in mixtures, thereby reducing the complexity of mass spectra obtained by MALDI MS. The LLE technique was optimized for rapid and sensitive in situ (on-target) sample preparation for MALDI MS analysis of proteins and peptides at low-picomole and subpicomole levels. Addition of MALDI matrix to the organic solvent enhanced the efficiency of the LLE-MALDI MS method for analysis of hydrophobic peptides and proteins. LLE-MALDI MS enabled the detection of the hydrophobic membrane protein bacteriorhodopsin as a component in a simple protein mixture. Peptide mixtures containing phosphorylated, glycosylated, or acylated peptides were successfully separated and analyzed by the in situ LLE-MALDI MS technique and demonstrate the potential of this method for enhanced separation and structural analysis of posttranslationally modified peptides in proteomics research.     

Separation and detection of phosphorylated and nonphosphorylated peptides in liquid chromatography-mass spectrometry using monolithic columns and acidic or alkaline mobile phases

Efficient chromatographic separation is a prerequisite for the sensitive analysis of complex peptide mixtures using liquid chromatography-mass spectrometry. This is especially true for the analysis of mixtures of unmodified and posttranslationally modified peptides, for example, phosphorylated peptides in the presence of their unmodified analogues. Applying monolithic capillary columns based on poly(styrene/divinylbenzene), the influence of acidic eluents based on trifluoroacetic and heptafluorobutyric acid as well as an alkaline eluent based on triethylamine-acetic acid (pH 9.2) on the separation of synthetic phosphopeptides was evaluated. Heptafluorobutyric acid offered the longest retention times and highest selectivities and, hence, the most effective separation. Application of the alkaline eluent in conjunction with detection in negative ion mode electrospray ionization mass spectrometry, on the other hand, allowed the detection of phosphorylated peptides with significantly lower limits of detection, as compared to acidic eluents in combination with detection in positive ion mode. Pairs of phosphorylated and nonphosphorylated synthetic peptides, ranging from 7- to 16-mers, as well as phosphorylated peptides form a tryptic protein digest could be separated both at acidic and alkaline pH. Utilizing a 60 x 0.20-mm-i.d. capillary column, the limit of detection in negative ion detection mode for a 4-fold phosphorylated peptide in a beta-casein digest was 10 fmol. Together with the capability for fast separation of protein digests, monolithic columns, thus, facilitate the effective and sensitive analysis of this important posttranslational modification.     

Development of a protein chip: a MS-based method for quantitation of protein expression and modification levels using an immunoaffinity approach

Protein chip technology permits analysis of the expression and modification status of numerous targeted proteins within a single experiment, mainly through the use of antibody-based microarrays. Despite recent improvements in these protein chips, their applications are still limited for a variety of reasons, which include technical challenges in fabrication of the antibody chips as well as the very low specificity achieved by current detection methods. We have developed a unique approach for relative and/or absolute quantitation of protein expression and modification based on the capture of epitope peptides on affinity beads, which can be used to develop a mass-spectrometry-based protein chip technology. This new method, which utilizes antibodies immobilized on beads for the capture of target peptides, instead of proteins, eliminates many of the problems previously associated with protein chips. We present here several proof-of-principle experiments examining model peptides by this technique. These experiments show that the method is capable of (i). detecting peptides bound to a single antibody bead, (ii). detecting peptides at low (fmol) levels, (iii). producing MS/MS data of suitable quality for protein identification via database searching or de novo sequencing, (iv). quantitating peptides affinity-bound to antibody beads, (v). specifically detecting target peptides in complex mixtures over wide dynamic ranges, and (vi) is compatible with a microarray format for high-throughput analysis. Because our novel method uses antibody beads instead of a derivatized capture surface, and peptides instead of proteins for affinity capture, it can overcome many of the pitfalls of previous protein chip fabrications. Therefore, this method offers an improved approach to protein chip technology that should prove useful for diagnostics and drug development applications.