Detection of phosphopeptides using Fe(III)-nitrilotriacetate complexes immobilized on a MALDI plate

Metal affinity complexes were chemically grafted onto the surface of gold matrix-assisted laser desorption/ionization (MALDI) plates by coupling a derivative of nitrilotriacetate (NTA) to immobilized poly(acrylic acid) (PAA) and subsequently forming the Fe(III)-NTA complex. The immobilized complexes can adsorb phosphorylated peptides preferentially from protein digests; deposition of digests on these surface-modified plates, followed by rinsing with an acetic acid solution, addition of matrix, and subsequent analysis by MALDI MS, resulted in mass spectra dominated by peaks corresponding to phosphopeptides. In the case of analyzing a tryptic digest of beta-casein, conventional MALDI MS revealed only one monophosphopeptide, while use of the Fe(III)-NTA-PAA-modified plate resulted in strong signals due to two additional tetraphosphorylated species. The diminution or elimination of signals due to nonphosphorylated species also greatly simplified the identification of phosphopeptides during analysis of ovalbumin digests and myoglobin digests spiked with an equimolar mixture of angiotensin and phosphoangiotensin. The matrix 2',4',6'-trihydroxyacetophenone mixed with diammonium hydrogen citrate proved to be much better than alpha-cyano-4-hydroxycinnamic acid for the detection of phosphorylated peptides from digests of beta-casein and ovalbumin.     

Capture of phosphopeptides using alpha-zirconium phosphate nanoplatelets

Alpha-zirconium phosphate nanoplatelets (alpha-ZrPN) were studied as a binding agent for phosphopeptides. Nanoplatelets of alpha-zirconium phosphate were incubated overnight with zirconium oxychloride, followed by centrifugation, and washed twice with water followed by an aqueous solution of 80% acetonitrile to form the binding agent. Alpha-ZrPN were able specifically to capture phosphoserine-containing peptides from a tryptic digest of a complex peptide mixture in which its abundance was only 0.05%. Alpha-ZrPN also bound peptides containing phosphothreonine and phosphotyrosine. The limit of detection for phosphopeptides is approximately 2 fmol, based on using matrix-assisted laser desorption/ionization mass spectrometry. Alpha-ZrPN were applied for the analysis of tryptic digests of mouse liver and leukemia cell phosphoproteomes and succeeded in identifying 158 phosphopeptides (209 phosphorylation sites) from 101 phosphoproteins in mouse liver lysate and 78 phosphopeptides (104 phosphorylation sites) from 59 phosphoproteins in leukemia cell extract. For these two tryptic digests, the alpha-ZrPN approach is able to capture more phosphopeptides than that obtained from TiO2 particles or from Fe(3+)-IMAC beads, but each method is able to bind some phosphopeptides that the others do not.     

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.     

Use of polymer-modified MALDI-MS probes to improve analyses of protein digests and DNA

The use of sample probe surfaces patterned with 200-microm-diameter spots of hydrophilic, charged polymers significantly enhances the analysis of protein digests and DNA by MALDI-MS. Selective adsorption on these polymer-modified surfaces allows collection of specific proteolytic peptides, while subsequent rinsing of the deposited sample removes contaminants. In the case of partially digested myoglobin, the mass spectrum obtained using a sample probe modified with polyanionic functionalities permits detection of 22 proteolytic fragments, while analysis using a stainless steel MALDI sample probe gives only 11 detectable fragments. Similarly, during the analysis of bovine serum albumin digests, the use of several different surface-modified MALDI sample probes increases sequence coverage from 61.3 to 74.5%. Detection of phosphorylated peptides can be quite challenging during analyses of phosphoprotein digests by MALDI-MS because these anionic proteolytic fragments have low ionization efficiencies. However, MALDI signals from the phosphorylated proteolytic fragments sometimes increase dramatically when using a sample probe surface modified by a polycation (polyethylenimine or poly(acrylic acid) complexed with Fe(3+)). The signal enhancement apparently occurs because the positive surface selectively binds the phosphorylated peptides. The use of patterned, polycationic surfaces also shows great promise for selective adsorption and decontamination of DNA samples; a simple water rinse diminishes or eliminates the formation of multi-ion adducts, thereby improving mass resolution during subsequent analysis by MALDI-MS.     

Mass spectrometry analysis of phosphopeptides after peptide carboxy group derivatization

A nearly 100% yield peptide carboxy group derivatization method was offered to largely enhance phosphopeptide ionization efficiency. This method, adopting 1-(2-pyrimidyl) piperazine (PP) as the derivatization reagent, shows several advantages such as good reproducibility, ease of handling, rapid reaction time, and no side reaction. PP derivatization improves the hydrophobicities, p I values, and gas-phase basicities of peptides especially those of phosphopeptides. In the matrix assisted laser desorption ionization (MALDI) source, the ionization efficiencies of four synthetic phosphopeptides were increased by 50-101 times while that of three nonphosphopeptides were 10-40-fold. In the electrospray ionization (ESI) source, PP-derivatized phosphopeptides also gave much higher ionization efficiency improvements than nonphosphopeptides. When this method was applied to much more complex mixtures, tryptic BSA digests spiked with one single phosphopeptide in different molar ratios, the signal intensity of this phosphopeptide always had the largest increment among all those peptides. Obviously, this easily manipulated as well as highly specific method provides a promising tool for high-throughput phosphoproteome research.     

Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns

Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel automated method for the enrichment of phosphopeptides from complex mixtures. The method employs a two-dimensional column setup, with titanium oxide-based solid-phase material (Titansphere) as the first dimension and reversed-phase material as the second dimension. Phosphopeptides are separated from nonphosphorylated peptides by trapping them under acidic conditions on a TiO(2) precolumn. Nonphosphorylated peptides break through and are trapped on a reversed-phase precolumn after which they are analyzed by nanoflow LC-ESI-MS/MS. Subsequently, phosphopeptides are desorbed from the TiO(2) column under alkaline conditions, reconcentrated onto the reversed-phase precolumn, and analyzed by nanoflow LC-ESI-MS/MS. The selectivity and practicality of using TiO(2) precolumns for trapping phosphopeptides are demonstrated via the analysis of a model peptide RKISASEF, in a 1:1 mixture of a non- and a monophosphorylated form. A sample of 125 fmol of the phosphorylated peptide could easily be isolated from the nonphosphorylated peptide with a recovery above 90%. In addition, proteolytic digests of three different autophosphorylation forms of the 153-kDa homodimeric cGMP-dependent protein kinase are analyzed. From proteolytic digests of the fully autophosphorylated protein at least eight phosphorylation sites are identified, including two previously uncharacterized sites, namely, Ser-26 and Ser-44. Ser-26 is characterized as a minor phosphorylation site in purified PKG samples, while Ser-44 is identified as a novel in vitro autophosphorylation target. These results clearly show that TiO(2) has strong affinity for phosphorylated peptides, and thus, we conclude that this material has a high potential in the field of phosphoproteomics.     

Selective enrichment and fractionation of phosphopeptides from peptide mixtures by isoelectric focusing after methyl esterification

We have developed a new strategy to enrich and fractionate phosphopeptides from peptide mixtures based on the difference in their isoelectric points (pIs) after methyl esterification. After isoelectric focusing (IEF) of a methylated tryptic digest of a mixture of alpha-S-casein and beta-casein, phosphopeptides were selectively enriched at acidic and neutral pHs while nonphosphopeptides left the focusing gel because their pIs are higher than the upper limit of the immobilized pH gradient. We wrote a web-based program, pIMethylation, to predict the pIs for peptides with and without methyl esterification. Theoretical calculations using pIMethylation indicated that methylated phosphopeptides and non-phosphopeptides can be grouped on the basis of the number of phosphate groups and basic residues in each peptide. Our IEF results were consistent with theoretical pIs of methylated peptides calculated by pIMethylation. We also showed that 2,6-dihydroxy-acetophenone is superior to 2,5-dihydroxybenzoic acid as a matrix for MALDI Q-TOF MS of methylated phosphopeptides in both positive and negative ion modes.     

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.     

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.     

Impulse-driven heated-droplet deposition interface for capillary and microbore LC-MALDI MS and MS/MS

An automated off-line liquid chromatography-matrix-assisted laser desorption ionization (LC-MALDI) interface capable of coupling both capillary and microbore LC separations with MALDI mass spectrometry (MS) and tandem mass spectrometry (MS/MS) has been developed. The interface is a combination of two concepts: analyte concentration from heated hanging droplets and impulse-driven droplet deposition of LC fractions onto a MALDI sample plate. At room temperature the interface allows the coupling of capillary LC separations (i.e., flow rate of <5 microL/min) with MALDI MS. With heating, it can be used to combine microbore LC operated at a relatively high flow rate of up to 50 microL/min with MALDI MS. The collected fractions can be analyzed by MALDI MS and MS/MS instruments, such as time-of-flight (TOF) and quadrupole-TOF MS. Performance of the interface was examined using several peptide and protein standards. It was shown that, using MALDI-TOF MS, [GLU1]-fibrinopeptide B could be detected with a total injection amount of 5 fmol to microbore LC. Chromatographic performance was also monitored. A peak width of 12 s at half-height for [GLU1]-fibrinopeptide B showed no evidence of band broadening due to the interface. The ability of the interface to mitigate ion suppression was studied using a mixture of 100 fmol of [GLU1]-fibrinopeptide B and 10 pmol of cytochrome c tryptic digest. Although fully suppressed under direct MALDI conditions, LC-MALDI analysis was able to detect the 100 fmol peptide with 10 s fraction collection. Finally, the ability to inject relatively large sample amounts to improve detectability of low-abundance peptides was illustrated in the analysis of phosphopeptides from alpha-casein tryptic digests. A digest loaded on column to 2.4 microg and analyzed by LC-MALDI MS/MS resulted in 82% sequence coverage and detection of all nine phosphoserine residues. It is concluded that, being able to handle both high- and low-flow LC separations, the impulse-driven heated-droplet interface provides the flexibility to carry out MALDI analysis of peptides and proteins depending on the information sought after, analysis speed, and sample size.     

Analysis of protein phosphorylation by hypothesis-driven multiple-stage mass spectrometry

We describe a strategy, which we term hypothesis-driven multiple-stage mass spectrometry (HMS-MS), for the sensitive detection and identification of phosphopeptides derived from enzymatic digests of phosphoproteins. In this strategy, we postulate that any or all of the potential sites of phosphorylation in a given protein may be phosphorylated. Using this assumption, we calculate the m/z values of all the corresponding singly charged phosphopeptide ions that could, in theory, be produced by the enzyme employed for proteolysis. We test ions at these m/z values for the presence of phosphoserine or phosphothreonine residues using tandem mass spectrometry (MS(2)) in a vacuum MALDI ion trap mass spectrometer, where the neutral loss of the elements of H(3)PO(4) (98 Da) provides a sensitive assay for the presence of phosphopeptides. Subsequent MS(3) analysis of the (M + H - 98)(+) peaks allows us to confirm or reject the hypotheses that the putative phosphopeptides are present in the sample. HMS-MS was successfully applied to the detection and identification of phosphopeptides from substrates of the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28, phosphorylated in vitro (Ipl1) and in vivo (Orc6), basing hypothesis formation on the minimal Cdk consensus phosphorylation motif Ser/Thr-Pro. The method was also used to find in vitro phosphopeptides from a domain of the Drosophila melanogaster protein PERIOD, hypothesizing possible phosphorylations of all Ser/Thr residues without assuming a consensus motif. Our results demonstrate that HMS-MS is a sensitive, highly specific tool for systematically surveying proteins for Ser/Thr phosphorylation, and represents a significant step toward our goal of comprehensive phosphorylation mapping.     

Characterization of protein phosphorylation by mass spectrometry using immobilized metal ion affinity chromatography with on-resin beta-elimination and Michael addition

A protocol combining immobilized metal ion affinity chromatography and beta-elimination with concurrent Michael addition has been developed for enhanced analysis of protein phosphorylation. Immobilized metal ion affinity chromatography was initially used to enrich for phosphorylated peptides. Beta-elimination, with or without concurrent Michael addition, was then subsequently used to simultaneously elute and derivatize phosphopeptides bound to the chromatography resin. Derivatization of the phosphate facilitated the precise determination of phosphorylation sites by MALDI-PSD/LIFT tandem mass spectrometry, avoiding complications due to ion suppression and phosphate lability in mass spectrometric analysis of phosphopeptides. Complementary use of immobilized metal ion affinity chromatography and beta-elimination with concurrent Michael addition in this manner circumvented several inherent disadvantages of the individual methods. In particular, (i) the protocol discriminated O-linked glycosylated peptides from phosphopeptides prior to beta-elimination/Michael addition and (ii) the elution of peptides from the chromatography resin as derivatized phosphopeptides distinguished them from unphosphorylated species that were also retained. The chemical derivatization of phosphopeptides greatly increased the information obtained during peptide sequencing by mass spectrometry. The combined protocol enabled the detection and sequencing of phosphopeptides from protein digests at low femtomole concentrations of initial sample and was employed to identify novel phosphorylation sites on the cell adhesion protein p120 catenin and the glycoprotein fetuin.     

Niobium(V) oxide (Nb2O5): application to phosphoproteomics

Proteomics-based analysis of signaling cascades relies on a growing suite of affinity resins and methods aimed at efficient enrichment of phosphorylated peptides from complex biological mixtures. Given the heterogeneity of phosphopeptides and the overlap in chemical properties between phospho- and unmodified peptides, it is likely that the use of multiple resins will provide the best combination of specificity, yield, and coverage for large-scale proteomics studies. Recently titanium and zirconium dioxides have been used successfully for enrichment of phosphopeptides. Here we report the first demonstration that niobium pentoxide (Nb 2O 5) provides for efficient enrichment and recovery ( approximately 50-100%) of phosphopeptides from simple mixtures and facilitates identification of several hundred putative sites of phosphorylation from cell lysate. Comparison of phosphorylated peptides identified from Nb 2O 5 and TiO 2 with sequences in the PhosphoELM database suggests a useful degree of divergence in the selectivity of these metal oxide resins. Collectively our data indicate that Nb 2O 5 provides efficient enrichment for phosphopeptides and offers a complementary approach for large-scale phosphoproteomics studies.     

Detection of phosphorylated peptides in proteomic analyses using microfluidic compact disk technology

A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.     

Integrated sample preparation and MALDI mass spectrometry on a microfluidic compact disk

High-throughput microfluidic processing of protein digests integrated with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on a compact disk (CD) is described. Centrifugal force moves liquid through multiple microstructures, each containing a 10-nL reversed-phase chromatography column. The CD enables parallel preparation of 96 samples with volumes ranging from one to several microliters. The peptides in the digests are concentrated, desalted, and subsequently eluted from the columns directly into MALDI target areas (200 x 400 microm) on the CD using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into the MALDI instrument for peptide mass fingerprinting and database identification at a routine sensitivity down to the 200-amol level. Detection of proteolytic peptides down to the 50-amol level is demonstrated. The success rate of the CD technology in protein identification is about twice that of the C(18) ZipTips and standard MALDI steel targets. The CDs are operated using robotics to transfer samples and reagents from microcontainers to the processing inlets on the disposable CD and spinning to control the movement of liquid through the microstructures.     

Phosphopeptide derivatization signatures to identify serine and threonine phosphorylated peptides by mass spectrometry

The development of rapid, global methods for monitoring states of protein phosphorylation would provide greater insight for understanding many fundamental biological processes. Current best practices use mass spectrometry (MS) to profile digests of purified proteins for evidence of phosphorylation. However, this approach is beset by inherent difficulties in both identifying phosphopeptides from within a complex mixture containing many other unmodified peptides and ionizing phosphopeptides in positive-ion MS. We have modified an approach that uses barium hydroxide to rapidly eliminate the phosphoryl group of serine and threonine modified amino acids, creating dehydroamino acids that are susceptible to nucleophilic derivatization. By derivatizing a protein digest with a mixture of two different alkanethiols, phosphopeptide-specific derivatives were readily distinguished by MS due to their characteristic ion-pair signature. The resulting tagged ion pairs accommodate simple and rapid screening for phosphopeptides in a protein digest, obviating the use of isotopically labeled samples for qualitative phosphopeptide detection. MALDI-MS is used in a first pass manner to detect derivatized phosphopeptides, while the remaining sample is available for tandem MS to reveal the site of derivatization and, thus, phosphorylation. We demonstrated the technique by identifying phosphopeptides from beta-casein and ovalbumin. The approach was further used to examine in vitro phosphorylation of recombinant human HSP22 by protein kinase C, revealing phosphorylation of Thr-63.     

A CE-MALDI interface based on the use of prestructured sample supports

We have developed an off-line coupling of capillary electrophoresis (CE) to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) based on CE fraction collection onto prestructured MALDI sample supports. Analyte carryover and detection sensitivity were investigated using a standard peptide mixture. Low femtomole amounts were detected, and no noticeable carryover was discovered. The performance of the method was evaluated with a mixture of tryptic digests of proteins from a human fetal brain cDNA expression library. The total number of identified peptides was increased from 47 to 211 when the CE-MALDI interface was used compared to direct MALDI-MS analysis. Sequence coverage with CE-MALDI was in the 25-60% range for the different proteins, corresponding to an increase of 1.3-4.9 times relative to that obtained with MALDI-MS of the crude mixture. Fractionation of sample components also facilitated protein identification by MALDI postsource decay analysis. Our initial results suggest this CE-MALDI interface can be used for the analysis of complex peptide mixtures isolated from biological tissues.     

Desorption/ionization on silicon nanowires

Dense arrays of single-crystal silicon nanowires (SiNWs) have been used as a platform for laser desorption/ionization mass spectrometry of small molecules, peptides, protein digests, and endogenous and xenobiotic metabolites in biofluids. Sensitivity down to the attomole level has been achieved on the nanowire surfaces by optimizing laser energy, surface chemistry, nanowire diameter, length, and growth orientation. An interesting feature of the nanowire surface is that it requires lower laser energy as compared to porous silicon and MALDI to desorb/ionize small molecules, therefore reducing background ion interference. Taking advantage of their high surface area and fluid wicking capabilities, SiNWs were used to perform chromatographic separation followed by mass analysis of the separated molecules providing a unique platform that can integrate separation and mass spectrometric detection on a single surface.     

Suppression of alpha-cyano-4-hydroxycinnamic acid matrix clusters and reduction of chemical noise in MALDI-TOF mass spectrometry

Progress in high-throughput MALDI-TOFMS analysis, especially in proteome applications, requires development of practical and efficient procedures for the preparation of proteins and peptides in a form suitable for high acquisition rates. These methods should improve successful identification of peptides, which depends on the signal intensity and the absence of interfering signals. Contamination of MALDI samples with alkali salts results in reduced MALDI peptide sensitivity and causes matrix cluster formation (widely reported for CHCA matrix) observed as signals dominating in the range below m/z 1200 in MALDI spectra. One way to remove these background signals, especially for concentrations of peptides lower than 10 fmol/microL, is to wash matrix/sample spots after peptide cocrystallization on the MALDI plate with deionized water prior to analysis. This method takes advantage of the low water solubility of the CHCA compared to its alkali salts. We report here that the application of some ammonium salt solutions, such as citrates and phosphates, instead of deionized water greatly improves the efficiency of this washing approach. Another way to reduce matrix cluster formation is to add ammonium salts as a part of the MALDI matrix. The best results were obtained with monoammonium phosphate, which successfully suppressed matrix clusters and improved sensitivity. Combining both of these approaches-the addition of ammonium salts in the CHCA matrix followed by one postcrystallization washing step with ammonium buffer-provided a substantial ( approximately 3-5-fold) improvement in the sensitivity of MALDI-MS detection compared to unwashed sample spots. This sample preparation method resulted in improved spectral quality and was essential for successful database searching for subnanomolar concentrations of protein digests.     

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.