Improved matrix-assisted laser desorption/ionization mass spectrometric analysis of tryptic hydrolysates of proteins following guanidination of lysine-containing peptides

Analysis of tryptic digests of proteins using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry commonly results in superior detection of arginine-containing peptides compared with lysine-containing counterparts. The effect is attributable in part to the greater stability of the arginine-containing peptide ions associated with the sequestration of the single ionizing proton on the arginine side-chain. Reaction of peptides with O-methylisourea resulted in conversion of lysine to homoarginine residues with consequent improved detection during MALDI-MS. Analysis of the underivatized tryptic digest of the yeast protein, enolase, revealed peptides representing 20% of the protein; the corresponding figure after derivatization was 46%.     

In vitro determination of the release kinetics of peptides and free amino acids during the digestion of food proteins

The kinetics of peptide release during in vitro digestion of 4 protein sources (casein, cod protein, soy protein, and gluten) were investigated. Samples were sequentially hydrolyzed with pepsin (30 min) and pancreatin (2, 4, or 6 h) in a dialysis cell with continuous removal of digestion products. Nondialyzed digests were fractionated by ion-exchange chromatography and ultrafiltration. Animal proteins were digested at a greater rate than plant proteins. Target amino acids of specific enzymes appeared more rapidly in the dialyzed fractions when compared to other amino acids. Throughout the hydrolysis, nondialyzed digests contained a higher proportion of peptide mixtures with basic-neutral properties. Except for gluten, peptide fractions with molecular weights that exceeded 10 kDa (basic-neutral, BN > 10) were rapidly hydrolyzed during the first 2 h of pancreatin digestion. The kinetics of release and the composition of peptide fractions were different when the protein sources were compared. The analysis of amino acids revealed that threonine and proline proportions were relatively high in BN > 10 and in peptide fractions with molecular weight between 10-1 kDa (BN 10-1), while tyrosine, phenylalanine, lysine, and arginine predominated in the low molecular weight (<1 kDa) fractions. More resistant peptides were generally rich in proline and glutamic acid. The role of in vitro digestion assays in dietary protein quality evaluation is discussed.     

Efficient enrichment and desalting of protein digests using magnetic mesocellular carbon foams in matrix-assisted laser desorption/ionization mass spectrometry

We demonstrate that magnetic mesocellular carbon foams (Mag-MCF-C) can be effectively used for enrichment and desalting of protein digests or peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The large mesocellular pores and surface area of Mag-MCF-C are likely to mainly contribute to high efficiency in enrichment and desalting of protein digests. The magnetic property of Mag-MCF-C enabled easy and simple enrichment and desalting process comprising adsorption, washing, and separation steps by using an external magnet. Following elution from Mag-MCF-C by using a matrix solution (CHCA in 70% ACN/0.1% TFA), the peptides were subjected to MALDI-MS analysis. As a result, MALDI mass spectra of peptides or tryptic protein digests were distinct even at a peptide concentration as low as 50 pM. The use of Mag-MCF-C resulted in significantly improved sequence coverage for protein identification when compared to other conventional methods. Mag-MCF-C will find applications in mass spectrometric analysis of low abundance peptides or protein digests with high sensitivity.     

Rapid determination of amino acid incorporation by stable isotope labeling with amino acids in cell culture (SILAC)

Stable isotope labeling with amino acids in cell culture (SILAC) has evolved to be a major technique for quantitative proteomics using cell cultures. We developed a rapid method to follow and determine the incorporation of arginine and lysine. Analysis of the heavy state is required to avoid quantification errors. Moreover, the mixture of light and heavy states can be exploited to normalize the protein amount for subsequent relative quantification experiments. Therefore, peptides from different cell lines were extracted with 0.1% trifluoroacetic acid and analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS). This analysis was highly reproducible and was performed in less than 2 h, significantly faster than other methods for the same purpose. Similar peptide mass profiles were obtained for human EBV-transformed B, Jurkat T, and HeLa cells as well as for mouse embryonic fibroblasts. Proteolytic fragments of 27 human proteins were identified with 56 peptides by MALDI-MS/MS and can be used as a database for these kinds of experiments. Sequencing revealed that the peptides were predominantly amino- and carboxy-terminal protein fragments displaying a specificity characteristic of the acidic proteases cathepsin D and E. Many of the identified peptides contained arginine and/or lysine, allowing determination of the incorporation rate of these amino acids. Furthermore, the rate of conversion of arginine into proline could be monitored easily.     

Multifunctional nanoparticles composite for MALDI-MS: Cd2+-doped carbon nanotubes with CdS nanoparticles as the matrix, preconcentrating and accelerating probes of microwave enzymatic digestion of peptides and proteins for direct MALDI-MS analysis

For the first time, we utilized multifunctional nanoparticles composite (NPs composite) for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis of peptides and proteins. Multiwalled carbon nanotubes doped with Cd(2+) ions and modified with cadmium sulfide NPs were synthesized by a chemical reduction method at room temperature. The multifunctional NPs composite applied for the analysis of peptides and microwave-digested proteins in the atmospheric pressure matrix-assisted laser desorption/ionization ion-trap and MALDI time-of-flight (TOF) mass spectrometry (MS) was successfully demonstrated. The maximum detection sensitivity for peptides in MALDI-MS was achieved by the adsorption of negatively charged peptides onto the surfaces of NP composite through electrostatic interactions. The optimal conditions of peptide mixtures were obtained at 20 min of incubation time using 1 mg of NPs composite when the pH of the sample solution was kept higher than the pI values of peptides. The potentiality of the NP composite in the preconcentration of peptides was compared with that of the individual NP by calculating the preconcentration factors (PF) and found that the NPs composite showed a 4-6 times of PF than the other NPs. In addition, the NPs composite was also applied as heat-absorbing materials for efficient microwave tryptic digestion of cytochrome c and lysozyme from milk protein in MALDI-TOF-MS analysis. We believe that the use of NPs composite technique would be an efficient and powerful preconcentrating tool for MALDI-MS for the study of proteome research.     

Enhanced responses in matrix-assisted laser desorption/ionization mass spectrometry of peptides derivatized with arginine via a C-terminal oxazolone

We have developed a novel method for enhancing the response of a peptide in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) by activating the C-terminal carboxyl group through an oxazolone with which is coupled an amine containing a functional group to help ionize the peptide. The reactions consist of dehydration with acetic anhydride to give an oxazolone, followed by aminolysis with an appropriate amino acid derivative such as arginine methyl ester. The MALDI signal of Ac-Tyr-Gly-Gly-Phe-Leu-Arg-OMe, thus converted from leucine-enkephalin, was detected while completely excluding the responses of arginine-deficient peptides coexisting in the reaction mixture. Some less intense peaks corresponding to a few sequential degradation products, also terminated with the arginine derivative, were also observed. The side-chain groups potentially that are reactive were conveniently protected by acetylation simultaneous with the C-terminal activation, and those that remained unprotected were reduced to virtually negligible proportions when the reaction was conducted in a peptide solution of concentration less than 1 mM. The greatly increased responses of such arginine-terminated peptides could possibly be exploited to discern the C-terminal tryptic peptide of a protein that is otherwise almost insensitive to MALDI-MS in general. The simplicity of the post-source decay spectrum of enkephalin derivatized by arginine methyl ester characteristically accentuated z- and b-type ions, and this should facilitate sequencing of such derivatized peptides. Remaining problems with practical applications of this approach are discussed.     

Ionic liquid matrices with phosphoric acid as matrix additive for the facilitated analysis of phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool for the analysis and characterization of protein phosphorylation on the peptide level. In this study, the applicability of ionic liquid matrices (ILM) formed by combination of the crystalline MALDI matrix 2,5-dihydroxybenzoic acid (DHB) with pyridine or n-butylamine was tested for the analysis of phosphopeptides. Low ionization efficiency in both positive and negative ion mode was observed in acid-free sample preparations. Upon addition of 0.1% trifluoroacetic acid (TFA), ion formation was increased, but analogously to the situation described earlier for pure DHB, best results were obtained upon use of 1% phosphoric acid as matrix additive. The samples prepared in this way were significantly more homogeneous than preparations with pure DHB, thus avoiding the need for time-consuming search for hot spots. Other characteristics like metastable fragmentation of phosphopeptides did not differ from that observed in classical preparations. The limits of detection for synthetic phosphopeptides and singly or multiply phosphorylated peptides from tryptic digests of alpha- and beta-casein were comparable with those obtained when using pure DHB; in some cases even higher signal intensities could be observed in the ILM. The use of ILM in combination with 1% phosphoric acid as matrix additive significantly facilitates analysis of phosphopeptides by MALDI-MS.     

Matrix-assisted laser desorption/ionisation mass spectrometric response factors of peptides generated using different proteolytic enzymes

Matrix-assisted laser desorption/ionisation (MALDI) mechanisms and the factors that influence the intensity of the ion signal in the mass spectrum remain imperfectly understood. In proteomics, it is often necessary to maximise the peptide response in the mass spectrum, especially for low abundant proteins or for proteolytic peptides of particular significance. We set out to determine which of the common proteolytic enzymes give rise to peptides with the best response factors under MALDI conditions. Standard proteins were enzymatically digested using four common proteases. We assessed relative response factors by coanalyzing the resulting digests. Thus, when tryptic peptides were added in equimolar quantities to their corresponding Asp-N, chymotrypsin and Glu-C digests, tryptic peptide signals were always predominant in the resulting MALDI mass spectra. Observable peaks attributable to non-tryptic peptides generally contained a terminal basic residue. It was proposed that a terminal basic residue has a disproportionate influence upon gas-phase basicity, and this hypothesis was supported by experiments with model isotopically labelled peptides. Experiments applying Cook's kinetic method showed that the peptide with a C-terminal arginine residue was more basic than the equivalent peptide with an N-terminal arginine, which was more basic than the peptide in which the arginine was mid-chain. Thus, the observation of the higher MALDI mass spectrometry response factors of tryptic peptides in comparison with peptides derived using other proteolytic enzymes corresponds with higher gas-phase basicities and may, along with other factors such as the complexity of the digest, influence the choice of enzyme in "bottom-up" proteomic experiments.     

Cooperative effect of factors governing molecular ion yields in desorption/ionization mass spectrometry

Factors governing the molecular ion yields of amino acids and peptides have been studied using fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) in positive-ion mode. The ion yields of protonated amino acids under FAB conditions are dependent on proton affinity (PA), hydrophobicity, and aromaticity of amino acids. Both PA and hydrophobicity contribute to an increase in the ion yields, while aromaticity contributes to a decrease. In MALDI, the ion yields increase linearly with the increase of PA of amino acids with the exception of lysine. In both FAB and MALDI experiments with peptides, the presence of arginine residues is essential for producing abundant protonated peptides. In FAB, the presence of aliphatic and hydrophobic amino acids (leucine and isoleucine) increases the ion yields of protonated peptides, while some hydrophilic amino acids (aspartic acid and asparagines) decrease the ion yields. The presence of two or more arginine residues does not give higher ion yields in FAB. In MALDI, the presence of aromatic amino acids (phenylalanine and tyrosine) enhances the signals for protonated peptides. Thus, physicochemical factors of individual amino acids cooperatively affect the ion yields of protonated amino acids and peptides. These factors governing the ion yields in FAB and MALDI affect two processes, desorption and ionization, that can be considered independently.     

低浓度甲醛对多肽和蛋白化学修饰的质谱研究

采用基质辅助激光解析电离飞行时间质谱( MALDI-TOF MS)和纳升电喷雾四极杆飞行时间串联质谱( Nano-ESI -QTOF MS)技术,以标准肽段和流感病毒基质蛋白酶切肽段为模型,研究了甲醛对蛋白质和多肽主链的修饰作用。采用与实际病毒灭活过程一致的实验条件(4℃,0.025%(V/V)福尔马林(37%(w/w)甲醛溶液)处理72 h),进行甲醛与多肽的化学反应。结果表明,在实验条件下,甲醛能与标准肽段N端的氨基反应生成羟甲基加合物,再发生缩合反应生成亚胺,形成+12 Da的产物。此外,甲醛还能与标准肽段中的精氨酸、赖氨酸的侧链发生反应,生成+12 Da的反应产物。对流感病毒基质蛋白的酶切肽段与甲醛的反应的质谱分析结果显示,多数的肽段都生成了+24 Da的产物,质量的增加来源于肽段N端氨基(+12 Da)和C端精氨酸或赖氨酸的侧链(+12 Da)的贡献。此外,还观察到有一个漏切位点的肽段生成了+36 Da的产物。本研究结果表明,在实验条件下,低浓度甲醛主要与肽段和蛋白的N 端氨基,以及精氨酸和赖氨酸侧链发生反应。本研究为分析低浓度甲醛与蛋白质的反应产物提供了有效的质谱分析方法和解谱依据。     

The relative influence of phosphorylation and methylation on responsiveness of peptides to MALDI and ESI mass spectrometry

Qualitative and quantitative analysis of post‐translational protein modifications by mass spectrometry is often hampered by changes in the ionization/detection efficiencies caused by amino acid modifications. This paper reports a comprehensive study of the influence of phosphorylation and methylation on the responsiveness of peptides to matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Using well‐characterized synthetic peptide mixtures consisting of modified peptides and their unmodified analogs, relative ionization/detection efficiencies of phosphorylated, monomethylated, and dimethylated peptides were determined. Our results clearly confirm that the ion yields are generally lower and the signal intensities are reduced with phosphopeptides than with their nonphosphorylated analogs and that this has to be taken into account in MALDI and ESI mass spectrometry. However, the average reduction of ion yield caused by phosphorylation is more pronounced with MALDI than with ESI. The unpredictable impact of phosphorylation does not depend on the hydrophobicity and net charge of the peptide, indicating that reliable quantification of phosphorylation by mass spectrometry requires the use of internal standards. In contrast to phosphorylation, mono‐ and dimethylated peptides frequently exhibit increased signal intensities in MALDI mass spectrometry (MALDI‐MS). Despite minor matrix‐dependent variability, MALDI methods are well suited for the sensitive detection of dimethylated arginine and lysine peptides. Mono‐ and dimethylation of the arginine guanidino group did not significantly influence the ionization efficiency of peptides in ESI‐MS. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of platinum nanoparticles as affinity probe and matrix for direct analysis of small biomolecules and microwave digested proteins using matrix-assisted laser desorption/ionization mass spectrometry

We report the use of platinum nanoparticles (PtNPs) for analysis of amino acids, peptides, proteins and microwave digested proteins (lysozyme and bovine serum albumin) without any tedious washing and separation procedures prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In the present study, PtNPs play three functions, such as matrix, affinity probe and acceleration of protein digestion by absorbing the microwave irradiation. Good signal intensity of the target molecules from the sample was obtained when laser energy, NPs concentration and incubation time were set to 35 μJ, 25 nM and 30 min, respectively. In addition, higher numbers of peptide sequence were obtained for microwave digested lysozyme protein using PtNPs as compared to previously reported methods for analysis of digested protein in MALDI-MS. Thus, the present method is a simple, rapid and one step preparation method for the analysis of amino acids, peptides, proteins and digested proteins in MALDI-TOF-MS without the need for any tedious purifications and washing procedures.     

Multifunctional ZrO2 nanoparticles and ZrO2-SiO2 nanorods for improved MALDI-MS analysis of cyclodextrins, peptides, and phosphoproteins

Multifunctional ZrO₂ nanoparticles (NPs) and ZrO₂-SiO₂ nanorods (NRs) have been successfully applied as the matrices for cyclodextrins and as affinity probes for enrichment of peptides (leucine-enkephalin, methionine-enkephalin and thiopeptide), phosphopeptides (from tryptic digestion products of β-casein) and phosphoproteins from complex samples (urine and milk) in atmospheric pressure matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and MALDI time-of-flight (TOF) MS. The results show that the ZrO₂ NPs and ZrO₂-SiO₂ NRs can interact with target molecules (cyclodextrins, peptides, and proteins), and the signal intensities of the analytes were significantly improved in MALDI-MS. The maximum signal intensities of the peptides were obtained at pH 4.5 using ZrO₂ NPs and ZrO₂-SiO₂ NRs as affinity probes. The limits of detection of the peptides were found to be 75-105 fmol for atmospheric pressure MALDI-MS and those of the cyclodextrins and β-casein were found to be 7.5-20 and 115-125 fmol, respectively, for MALDI-TOF-MS. In addition, these nanomaterials can be applied as the matrices for the analysis of cyclodextrins in urine samples by MALDI-TOF-MS. ZrO₂ NPs and ZrO₂-SiO₂ NRs efficiently served as electrostatic probes for peptide mixtures and milk proteins because 2–11 times signal enhancement can be achieved compared with use of conventional organic matrices. Moreover, we have successfully demonstrated that the ZrO₂ NPs can be effectively applied for enrichment of phosphopeptides from tryptic digestion of β-casein. Comparing ZrO₂ NPs with ZrO₂-SiO₂ NRs, we found that ZrO₂ NPs exhibited better affinity towards phosphopeptides than ZrO₂-SiO₂ NRs. Furthermore, the ZrO₂ and ZrO₂-SiO₂ nanomaterials could be used to concentrate trace amounts of peptides/proteins from aqueous solutions without tedious washing procedures. This approach is a simple, straightforward, separation-and washing-free approach for MALDI-MS analysis of cyclodextrins, peptides, proteins, and tryptic digestion products of phosphoproteins.      

Selective isolation-detection of two different positively charged peptides groups by strong cation exchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry: application to proteomics studies

We report here a procedure for the independent analysis of two groups of peptides by liquid chromatography-matrix-assisted laser desorption/ionization mass spectrometry (LC-MALDI MS/MS), using a selective isolation-detection procedure. In this procedure all primary amino groups of tryptic peptides derived from mouse liver proteins are blocked, restricting their positive charge, at acidic pH, to the presence of histidine and arginine residues. After strong cation exchange chromatography, multiply charged peptides (R + H > 1) are retained on the column and separated with high selectivity from singly (R + H = 1) and neutral peptides (R + H = 0) which are together collected in the flow-through. Using LC-MALDI-MS/MS analysis, the retained fraction displayed a 94% of enrichment of multiply charged peptides while in the flow-through; peptides with at least one arginine or histidine residue were exclusively identified, which suggests that MS detection in this fraction is restricted only to those peptides with ionizable side chains, arginine and histidine amino acids.     

Signature-peptide approach to detecting proteins in complex mixtures

The objective of the work presented in this paper was to test the concept that tryptic peptides may be used as analytical surrogates of the protein from which they were derived. Proteins in complex mixtures were digested with trypsin and classes of peptide fragments selected by affinity chromatography, lectin columns were used in this case. Affinity selected peptide mixtures were directly transferred to a high-resolution reversed-phase chromatography column and further resolved into fractions that were collected and subjected to matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The presence of specific proteins was determined by identification of signature peptides in the mass spectra. Data are also presented that suggest proteins may be quantified as their signature peptides by using isotopically labeled internal standards. Isotope ratios of peptides were determined by MALDI mass spectrometry and used to determine the concentration of a peptide relative to that of the labeled internal standard. Peptides in tryptic digests were labeled by acetylation with acetyl N-hydroxysuccinimide while internal standard peptides were labeled with the trideuteroacetylated analogue. Advantages of this approach are that (i) it is easier to separate peptides than proteins, (ii) native structure of the protein does not have to be maintained during the analysis, (iii) structural variants do not interfere and (iv) putative proteins suggested from DNA databases can be recognized by using a signature peptide probe.     

Interactions between sodium dodecyl sulfate micelles and peptides during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of proteolytic digests

Although sodium dodecyl sulfate (SDS) is routinely used as a denaturing agent for proteins, its presence is highly detrimental on the analysis of peptides and proteins by mass spectrometry. It has been found, however, that when SDS is present in concentrations near to or above its critical micelle concentration (CMC), improvements in the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of peptide mixtures or hydrophobic proteins are obtained. To elucidate possible explanations for such improvements, here we have undertaken a study examining the effect of SDS micelles on peptide mixtures. Fluorescently labeled peptides were used as probes to determine whether hydrophobic or hydrophilic peptides interact exclusively with SDS micelles. In addition, four globular proteins were digested with trypsin and then various amounts of SDS were added before MALDI mass spectrometry. To examine the role of mixture complexity on the mass spectral results, the tryptic digest of bovine serum albumin was also fractionated according to hydrophobicity before SDS treatment. Results from these experiments suggest that micelle-peptide interactions increase peptide-matrix cocrystallization irrespective of analyte hydrophobicity. As these studies were performed using the dried-droplet method of sample spotting, the presence of micelles is also hypothesized to reduce Marangoni effects during the crystallization process.     

Derivatization procedures to facilitate de novo sequencing of lysine-terminated tryptic peptides using postsource decay matrix-assisted laser desorption/ionization mass spectrometry

Guanidination of the epsilon-amino group of lysine-terminated tryptic peptides can be accomplished selectively in one step with O-methylisourea hydrogen sulfate. This reaction converts lysine residues into more basic homoarginine residues. It also protects the epsilon-amino groups against unwanted reaction with sulfonation reagents, which can then be used to selectively modify the N-termini of tryptic peptides. The combined reactions convert lysine-terminated tryptic peptides into modified peptides that are suitable for de novo sequencing by postsource decay matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The guanidination reaction is very pH dependent. Product yields and reaction kinetics were studied in aqueous solution using either NaOH or diisopropylethylamine as the base. Methods are reported for derivatizing and sequencing lysine-terminated tryptic peptides at low pmole levels. The postsource decay (PSD) MALDI tandem mass spectra of a model peptide (VGGYGYGAK), the homoarginine analog and the sulfonated homoarginine analog are compared. These spectra show the influence that each chemical modification has on the peptide fragmentation pattern. Finally, we demonstrate that definitive protein identifications can be achieved by PSD MALDI sequencing of derivatized peptides obtained from solution digests of model proteins and from in-gel digests of 2D-gel separated proteins.     

Derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate for enhancing the ionization yield of small peptides and glycopeptides in matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The characterization of glycosylation in proteins by mass spectrometry (MS) is often impeded by strong suppression of ionization of glycopeptides in the presence of non-glycosylated peptides. Glycopeptides with a large carbohydrate part and a short peptide backbone are particularly affected by this problem. To meet the goal of generating mass spectra exhibiting glycopeptide coverages as complete as possible, derivatization of glycopeptides offers a practical way to increase their ionization yield. This paper investigated derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) which is a rapid labeling technique commonly used for fluorescence detection in high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). As test samples we used peptides and glycopeptides obtained by enzymatic digestion of three different glycoproteins, i.e., human antithrombin, chicken ovalbumin, and bovine alpha1-acid-glycoprotein. It was found that AQC derivatization resulted in strongly increased signal intensities when analyzing small peptides and glycopeptides by matrix-assisted laser desorption/ionization (MALDI)-MS. For these compounds the limit of detection could be reduced to low fmol amounts. Without derivatization only glycopeptides containing large peptide backbones were detected by MALDI-MS. This effect was even significant when glycopeptides were pre-separated and enriched by means of lectin affinity chromatography before MALDI-MS analysis and when using electrospray ionization (ESI). This labeling method, applied in combination with MS detection for the first time, was found to be well suited for the enhancement of detection sensitivity for small glycopeptides in MALDI-MS analysis and thus for reducing the need for pre-separation steps.     

An evaluation of the utility of in vacuo methylation for mass-spectrometry-based analyses of peptides

In vacuo trimethylation of the N-terminus of a lyophilized peptide with methyl iodide was previously reported to enhance the peptide's signal in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and to suppress alkali adduct formation in electrospray ionization mass spectrometry (ESI-MS). Both the signal enhancement and alkali adduct suppression observed for methylated peptides are believed to be due to the permanent positive charge on the N-terminus of the peptide resulting from the formation of a quaternary ammonium moiety. The present work evaluates the general utility of the in vacuo methylation procedure for the MS analysis of peptides, and specifically addresses the issue of whether the methylation of nucleophilic sites other than the N-terminal amine affects the MALDI signal of modified peptides. This work establishes that, although certain side-chain modifications are inevitable using present reaction conditions, the derivatization leads to significant MALDI-MS signal improvement. The experimental results demonstrate that the N-terminal trimethylammonium derivatives of peptides exhibit MALDI signals comparable to or exceeding those of arginine-containing standards such as angiotensin I. The advantages and limitations of the in vacuo methylation procedure are discussed.     

Use of the arginine-specific butanedione/phenylboronic acid tag for analysis of peptides and protein digests using matrix-assisted laser desorption/ionization mass spectrometry

We have applied an arginine-specific labeling technique to the study of peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The reaction converts the guanidine group of the arginine side chain by reacting it with 2,3-butanedione and an arylboronic acid. Despite the general chemical lability of the tag under acidic conditions, it was possible to employ acidic matrices like alpha-cyano-4-hydroxycinnamic acid without adverse effects, using the thin-layer technique for preparation. After optimizing the method using arginine-containing model peptides--for which sensitivity down to the low fmol range was demonstrated--the procedure was applied to enzymatic digests of several model proteins in solution and to protein spots in gels obtained by two-dimensional electrophoretic separation of cell lysate samples. Information on the presence of arginine in peptides can be easily obtained from the mass spectra by the characteristic mass shift and the isotope pattern resulting from the incorporation of boron. This information might serve as a valuable additional search constraint for achieving a higher degree of confidence for protein identification by peptide mass fingerprinting.