De novo analysis of protein N-terminal sequence utilizing MALDI signal enhancing derivatization with Br signature

De novo analysis of protein N-terminal sequence is important for identification of N-terminal proteolytic processing such as N-terminal methionine or signal peptide removal, or for the genome annotation of uncharacterized proteins. We introduce a de novo sequencing method of protein N terminus utilizing matrix-assisted laser desorption/ionization (MALDI) signal enhancing picolinamidination with bromine isotopic tag incorporated to the N terminus. The doublet signature of bromine in the tandem mass (MS/MS) spectrum distinguished N-terminal ion series from C-terminal ion series, facilitating de novo N-terminal sequencing of protein. The dual advantage of MALDI signal enhancement by the basic picolinamidine and b-ion selection aided by Br signature is demonstrated using a variety of peptides. The N-terminal sequences of myoglobin and hemoglobin as model proteins were determined by incorporating the Br tag to the N terminus of the proteins and obtaining a series of b-ions with Br signature by MS/MS analysis after chymotryptic digestion of the tagged proteins. The N-terminal peptide was selected for MS/MS analysis from the chymotryptic digest based on the Br signature in the mass spectrum. Identification of phosphorylation site as well as N-terminal sequencing of a phosphopeptide was straightforward.     

Matrix-assisted laser desorption/ionization signal enhancement of peptides by picolinamidination of amino groups

Picolinamidination of amino groups in peptides was carried out using ethyl picolinimidate tetrafluoroborate synthesized from picolinamide and triethyloxonium tetrafluoroborate. The N-terminal amino group as well as the epsilon-amino group of lysine were derivatized. The matrix-assisted laser desorption/ionization (MALDI) signal of a peptide was enhanced 20-35-fold upon picolinamidination depending on the number of amino groups derivatized. The signal enhancement effect is much higher than that of acetamidination or guanidination previously reported. Improved protein identification by mass mapping of the derivatized peptides was demonstrated.     

A binary matrix for improved detection of phosphopeptides in matrix‐assisted laser desorption/ionization mass spectrometry

Application of matrix‐assisted laser‐desorption/ionization mass spectrometry (MALDI MS) to analysis and characterization of phosphopeptides in peptide mixtures may have a limitation, because of the lower ionizing efficiency of phosphopeptides than nonphosphorylated peptides in MALDI MS. In this work, a binary matrix that consists of two conventional matrices of 3‐hydroxypicolinic acid (3‐HPA) and α‐cyano‐4‐hydroxycinnamic acid (CCA) was tested for phosphopeptide analysis. 3‐HPA and CCA were found to be hot matrices, and 3‐HPA not as good as CCA and 2,5‐dihydroxybenzoic acid (DHB) for peptide analysis. However, the presence of 3‐HPA in the CCA solution with a volume ratio of 1:1 could significantly enhance ion signals for phosphopeptides in both positive‐ion and negative‐ion detection modes compared with the use of pure CCA or DHB, the most common phosphopeptide matrices. Higher signal intensities of phosphopeptides could be obtained with lower laser power using the binary matrix. Neutral loss of the phosphate group (?80 Da) and phosphoric acid (?98 Da) from the phosphorylated‐residue‐containing peptide ions with the binary matrix was decreased compared with CCA alone. In addition, since the crystal shape prepared with the binary matrix was more homogeneous than that prepared with DHB, searching for ‘sweet’ spots can be avoided. The sensitivity to detect singly or doubly phosphorylated peptides in peptide mixtures was higher than that obtained with pure CCA and as good as that obtained using DHB. We also used the binary matrix to detect the in‐solution tryptic digest of the crude casein extracted from commercially available low fat milk sample, and found six phosphopeptides to match the digestion products of casein, based on mass‐to‐charge values and LIFT TOF‐TOF spectra. Copyright © 2009 John Wiley & Sons, Ltd.     

Glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the O‐PS of Vibrio cholerae O1, serotype Ogawa,and BSA revealed by matrix‐assisted laser desorption–ionization tandem mass spectrometry

We present the MALDI‐TOF/TOF‐MS analyses of various hapten–bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer‐equipped, terminal monosaccharide of the O‐specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten–BSA neoglycoconjugates with different hapten–BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI‐TOF/TOF‐MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI‐TOF/TOF‐MS/MS of the glycated peptides. The product‐ion scans of the protonated molecules were carried out with a MALDI‐TOF/TOF‐MS/MS tandem mass spectrometer equipped with a high‐collision energy cell. The high‐energy collision‐induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y‐series product ions was very useful for the sequencing of various peptides. The series of a‐ and b‐product ions confirmed the sequence of the conjugated peptides. Copyright © 2010 John Wiley & Sons, Ltd.     

The specific isolation of C‐terminal peptides of proteins through a transamination reaction and its advantage for introducing functional groups into the peptide

A novel method for isolating C‐terminal peptides from proteolytic digests of proteins was developed. Proteins were digested with lysyl endopeptidase (LysC) and applied to metal‐ion‐catalyzed transamination reactions. This reaction enabled the selective conversion of an Nα‐amino group to a carbonyl group. Subsequent incubation with p‐phenylenediisothiocyanate (DITC) glass effectively scavenged the lysine‐containing N‐terminus and internal peptides. The obtained C‐terminal peptide is open to modification with reagents having virtually any type of functionality owing to the reactive α‐ketocarbonyl group. In this report, 2,4‐dinitrophenylhydrazine (DNPH) was used as an example of a nucleophile to the carbonyl group. The isolated C‐terminal peptide was modified with DNPH, which exhibited signal enhancement, and was sequenced by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Copyright © 2009 John Wiley & Sons, Ltd.     

Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro‐heterogeneity) and evaluate the molar site occupancy (macro‐heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N‐glycans was chemically synthesised by solid‐phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N‐acetylglucosamine‐linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well‐defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI‐IT, ESI‐Q‐TOF, MALDI‐TOF, ESI/MALDI‐FT‐ICR‐MS). Depending on the ion source/mass analyser, glycopeptides carrying complex‐type N‐glycans exhibited clearly lower signal strengths (10–50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano‐ESI and medium‐pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro‐heterogeneity and macro‐heterogeneity by label‐free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Characterization of braun’s lipoprotein and determination of its attachment sites to peptidoglycan by 252Cf-PD and MALDI time-of-flight mass spectrometry

A strategy for the characterization of bacterial lipoprotein-in this case Braun’s lipoprotein (an outer membrane 7-ku lipoprotein) isolated from Escherichia coli—is described by time-of-flight mass spectrometric (TOF/MS) techniques [²⁵²Cf plasma desorption (PD) TOF/MS and matrix-assisted laser desorption-ionization (MALDI) TOF/MS]. Covalent linkage of lipid at the N-terminal cysteine (posttranslationally modified to a S-[2,3-bis(acyloxy)-propyl]-N-acylcysteine) and, therefore, strict insolubility in aqueous solution constitute common features for this class of proteins. Relative molecular mass determination of the major molecular species of Braun’s lipoprotein was obtained by selection of an appropriate mixture of organic solvents compatible with matrix/support materials useful for the mass spectrometric techniques applied. Minor components of this lipoprotein that differ only in the fatty acid composition of the lipid anchor were detected by PD TOF/MS after enzymatic release of the extremely hydrophobic N-terminal amino acid followed by selective extraction with chloroform. Part of the primary sequence of this lipoprotein was confirmed based on peptide fragment ions observed in the positive ion PD mass spectra of cyanogen bromide-generated peptide fragments that had been isolated previously by reverse phase high-performance liquid chromatography (HPLC). Peptidoglycan fragments that represent the attachment sites of lipoprotein to peptidoglycan were enzymatically released, separated by reverse phase HPLC, and finally characterized by time-of-flight mass spectrometric techniques (²⁵²Cf-PD TOF/MS, MALDI TOF/MS). The results obtained with both techniques differed only in the better sensitivity obtained with MALDI TOF/MS, which consumed a factor of 100 to 1000 less material than with PD TOF/MS.     

Modeling bacteriophage amplification as a predictive tool for optimized MALDI‐TOF MS‐based bacterial detection

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) is a valuable tool for rapid bacterial detection and identification but is limited by the need for relatively high cell count samples, which have been grown under strictly controlled conditions. These requirements can be eliminated by the natural infection of a viable bacterial species of interest with a host‐specific phage. This produces a rapid increase in phage protein concentrations in comparison to bacterial concentrations, which can in turn be exploited as a method for signal amplification during MALDI‐TOF MS. One drawback to this approach is the requirement for repetitive, time‐consuming sample preparation and analysis applied over the course of a phage infection to monitor phage concentrations as a function of time to determine the MALDI‐TOF MS detection limit. To reduce the requirement for repeated preparation and analysis, a modified phage therapy model was investigated as a means for predicting the time during a given phage infection when a detectable signal would occur. The modified model used a series of three differential equations composed of predetermined experimental parameters including phage burst size and burst time to predict progeny phage concentrations as a function of time. Using Yersinia pestis with plague diagnostic phage ?A1122 and Escherichia coli with phage MS2 as two separate, well‐characterized model phage–host pairs, we conducted in silico modeling of the infection process and compared it with experimental infections monitored in real time by MALDI‐TOF MS. Significant agreement between mathematically calculated phage growth curves and those experimentally obtained by MALDI‐TOF MS was observed, thus verifying this method's utility for significant time and labor reduction. Copyright © 2012 John Wiley & Sons, Ltd.     

De novo sequence analysis and intact mass measurements for characterization of phycocyanin subunit isoforms from the blue‐green alga Aphanizomenon flos‐aquae

In this work, partial characterization of the primary structure of phycocyanin from the cyanobacterium Aphanizomenon flos‐aquae (AFA) was achieved by mass spectrometry de novo sequencing with the aid of chemical derivatization. Combining N‐terminal sulfonation of tryptic peptides by 4‐sulfophenyl isothiocyanate (SPITC) and MALDI‐TOF/TOF analyses, facilitated the acquisition of sequence information for AFA phycocyanin subunits. In fact, SPITC‐derivatized peptides underwent facile fragmentation, predominantly resulting in y‐series ions in the MS/MS spectra and often exhibiting uninterrupted sequences of 20 or more amino acid residues. This strategy allowed us to carry out peptide fragment fingerprinting and de novo sequencing of several peptides belonging to both α‐ and β‐phycocyanin polypeptides, obtaining a sequence coverage of 67% and 75%, respectively. The presence of different isoforms of phycocyanin subunits was also revealed; subsequently Intact Mass Measurements (IMMs) by both MALDI‐ and ESI‐MS supported the detection of these protein isoforms. Finally, we discuss the evolutionary importance of phycocyanin isoforms in cyanobacteria, suggesting the possible use of the phycocyanin operon for a correct taxonomic identity of this species. Copyright © 2008 John Wiley & Sons, Ltd.     

Sequence dependent fragmentation of peptides generated by MALDI quadrupole time-of-flight (MALDI Q-TOF) mass spectrometry and its implications for protein identification

A study has been undertaken to evaluate the usefulness of MALDI Q-TOF data for protein identification. The comparison of MS data of protein digests obtained on a conventional MALDI TOF instrument to the MS data from the MALDI Q-TOF reveal peptide patterns with similar intensity ratios. However, comparison of MS/MS Q-TOF data produced by nanoelectrospray versus MALDI reveals striking differences. Peptide fragment ions obtained from doubly charged precursors produced by nanoelectrospray are mainly y-type ions with some b-ions in the lower mass range. In contrast, peptide fragment ions produced from the singly charged ions originating from the MALDI source are a mixture of y-, b- and a-ions accompanied by ions resulting from neutral loss of ammonia or water. The ratio and intensity of these fragment ions is found to be strongly sequence dependent for MALDI generated ions. The singly charged peptides generated by MALDI show a preferential cleavage of the C-terminal bond of acidic residues aspartic and glutamic acid and the N-terminal bond of proline. This preferential cleavage can be explained by the mobile proton model and is present in peptides that contain both arginine and an acidic amino acid. The MALDI Q-TOF MS/MS data of 24 out of 26 proteolytic peptides produced by trypsin or Asp-N digestions were successfully used for protein identification via database searching, thus indicating the general usefulness of the data for protein identification. De novo sequencing using a mixture of 160/18O water during digestion has been explored and de novo sequences for a number of peptides have been obtained.     

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectroscopic analysis of telechelic polythiourethanes obtained by the cationic ring‐opening polymerization of six‐membered cyclic thiourethane

A matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectroscopy analysis of polythiourethanes obtained by the cationic ring‐opening polymerization of a six‐membered cyclic thiourethane [3‐benzyltetrahydro‐1,3‐oxazine‐2‐thione (BTOT)] is described. A MALDI‐TOF mass spectrum of a polymer obtained by the polymerization of BTOT with boron trifluoride etherate (BF₃OEt₂) as the initiator in nitrobenzene at 50 °C for 24 h followed by an end‐capping reaction with diethyldithiocarbamic acid diethylammonium salt showed a series of well‐resolved signals that were assignable to polythiourethanes possessing an amino group at the initiating end and a diethyldithiocarbamate group at the terminating end. In comparison with the MALDI‐TOF mass spectra of polymers obtained by polymerization with trifluoromethanesulfonic acid or methyl trifluoromethanesulfonate, the plausible initiating species in the polymerization with BF₃OEt₂ was estimated to be a proton, which successively eliminated carbonyl sulfide to produce a secondary amine group at the initiating end. The secondary amine group in the obtained telechelic polymer was converted to a tertiary amine group by a reaction with benzyl bromide in the presence of triethylamine, and this was confirmed by MALDI‐TOF mass spectroscopy. Furthermore, a telechelic polymer with a pyrrole end group was successfully synthesized by the end‐capping reaction of the growing species in the polymerization of BTOT with sodium 1‐pyrrolecarbodithioate. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 4281–4289, 2006     

Block length determination of the block copolymer mPEG‐b‐PS using MALDI‐TOF MS/MS

The molar mass determination of block copolymers, in particular amphiphilic block copolymers, has been challenging with chromatographic techniques. Therefore, methoxy poly(ethylene glycol)‐b‐poly(styrene) (mPEG‐b‐PS) was synthesized by atom transfer radical polymerization (ATRP) and characterized in detail not only by conventional chromatographic techniques, such as size exclusion chromatography (SEC), but also by matrix‐assisted laser/desorption ionization tandem mass spectrometry (MALDI‐TOF MS/MS). As expected, different molar mass values were obtained in the SEC measurements depending on the calibration standards (either PEG or PS). In contrast, MALDI‐TOF MS/MS analysis allowed the molar mass determination of each block, by the scission of the weakest point between the PEG and PS block. Thus, fragments of the individual blocks could be obtained. The PEG block showed a depolymerization reaction, while for the PS block fragments were obtained in the monomeric, dimeric, and trimeric regions as a result of multiple chain scissions. The block length of PEG and PS could be calculated from the fragments recorded in the MALDI‐TOF MS/MS spectrum. Furthermore, the assignment of the substructures of the individual blocks acquired by MALDI‐TOF MS/MS was accomplished with the help of the fragments that were obtained from the corresponding homopolymers. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Sodiation as a tool for enhancing the diagnostic value of MALDI‐TOF/TOF‐MS spectra of complex astaxanthin ester mixtures from Haematococcus pluvialis

The microalga Haematococcus pluvialis produces the pigment astaxanthin mainly in esterified form with a multitude of fatty acids, which results in a complex mixture of carotenol mono‐ and diesters. For rapid fingerprinting of these esters, matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF/TOF‐MS) might be an alternative to traditional chromatographic separation combined with MS. Investigation of ionization and fragmentation of astaxanthin mono‐ and diester palmitate standards in MALDI‐TOF/TOF‐MS showed that sodium adduct parent masses [M + Na]⁺ gave much simpler MS² spectra than radical / protonated [M]^+● / [M + H]⁺ parents. [M + Na]⁺ fragments yielded diagnostic polyene‐specific eliminations and fatty acid neutral losses, whereas [M]^+● / [M + H]⁺ fragmentation resulted in a multitude of non‐diagnostic daughters. For diesters, a benzonium fragment, formed by polyene elimination, was required for identification of the second fatty acid attached to the astaxanthin backbone. Parents were forced into [M + Na]⁺ ionization by addition of sodium acetate, and best signal‐to‐noise ratios were obtained in the 0.1 to 1.0 mM range. This method was applied to fingerprinting astaxanthin esters in a crude H. pluvialis extract. Prior to MALDI‐TOF/TOF‐MS, the extract was fractionated by normal phase Flash chromatography to obtain fractions enriched in mono‐ and diesters and to remove pheophytin a, which compromised monoester signals. All 12 types of all‐trans esterified esters found in LC were identified with MALDI‐TOF/TOF‐MS, with the exception of two minor monoesters. Copyright © 2013 John Wiley & Sons, Ltd.     

Application of electrospray mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry for molecular weight assignment of peptides in complex mixtures

Electrospray mass spectrometry (ES/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) were used to provide mass spectra from seven elapid snake venoms. Spectral interpretation was much simpler for MALDI/TOF/MS. ES/MS proved more useful for the provision of molecular weight data for very closely related peptides, but suppression of higher molecular weight compounds was seen to occur during flow injection analysis. MALDI/TOF/MS proved useful for providing a complete picture of the venom, but the low resolution led to obscuring of major ions, and the mass accuracy was poorer for known peptides. Suppression also occurred during MALDI/TOF/MS but could be overcome using alternative matrices because the spectra were very dependent on the choice of matrix. ES/MS and MALDI/TOF/MS provide complementary and confirmatory information such that for the anal sis of complex peptide mixtures (snake venoms), the use of both techniques is desirable.     

磷酸铵盐作为基质添加物促进磷酸化肽在MALDI-MS分析中的离子化效率

本文建立了以磷酸铵盐为添加剂的基质新系统,增强了磷酸化肽在MALDI正离子模式下的离子化。系统地考察了不同的磷酸盐以及不同的盐浓度对磷酸化肽离子化效率的影响。考察了两种适合于磷酸化肽离子化的基质类型2,5-二羟基苯甲酸和2,4,6-三羟基苯乙酮。用2,5-二羟基苯甲酸作为基质时,当加入10 mM 磷酸氢二铵时,磷酸化蛋白质β-casein的磷酸肽 48FQ[pS]EEQQQTEDELQDK63的离子化效率可以增强5-8倍,当加入10 mM磷酸二氢胺时,磷酸肽的离子化效率可以增强3-4倍。用2,4,6-三羟基苯乙酮作为基质时,当加入5mM磷酸氢二铵时,磷酸化肽的离子化效率比文献报道的最有利于磷酸化肽离子化的基质体系增强了2倍。并探讨了铵根离子和磷酸根离子促进磷酸化肽在MALDI的正离子模式下离子化效率的机理。     

Phosphorylated serine and threonine residues promote site‐specific fragmentation of singly charged,arginine‐containing peptide ions

In order to investigate gas‐phase fragmentation reactions of phosphorylated peptide ions, matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass (MS/MS) spectra were recorded from synthetic phosphopeptides and from phosphopeptides isolated from natural sources. MALDI‐TOF/TOF (TOF: time‐of‐flight) spectra of synthetic arginine‐containing phosphopeptides revealed a significant increase of y ions resulting from bond cleavages on the C‐terminal side of phosphothreonine or phosphoserine. The same effect was found in ESI‐MS/MS spectra recorded from the singly charged but not from the doubly charged ions of these phosphopeptides. ESI‐MS/MS spectra of doubly charged phosphopeptides containing two arginine residues support the following general fragmentation rule: Increased amide bond cleavage on the C‐terminal side of phosphorylated serines or threonines mainly occurs in peptide ions which do not contain mobile protons. In MALDI‐TOF/TOF spectra of phosphopeptides displaying N‐terminal fragment ions, abundant b–H₃PO₄ ions resulting from the enhanced dissociation of the pSer/pThr–X bond were detected (X denotes amino acids). Cleavages at phosphoamino acids were found to be particularly predominant in spectra of phosphopeptides containing pSer/pThr–Pro bonds. A quantitative evaluation of a larger set of MALDI‐TOF/TOF spectra recorded from phosphopeptides indicated that phosphoserine residues in arginine‐containing peptides increase the signal intensities of the respective y ions by almost a factor of 3. A less pronounced cleavage‐enhancing effect was observed in some lysine‐containing phosphopeptides without arginine. The proposed peptide fragmentation pathways involve a nucleophilic attack by phosphate oxygen on the carbon center of the peptide backbone amide, which eventually leads to cleavage of the amide bond. Copyright © 2009 John Wiley & Sons, Ltd.     

Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for recovery, detection and sequencing of phosphopeptides

An integrated analytical strategy for enrichment, detection and sequencing of phosphorylated peptides by matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) is reported. o-Phosphoric acid was found to enhance phosphopeptide ion signals in MALDI-MS when used as the acid dopant in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. The effect was largest for multiply phosphorylated peptides, which exhibited an up to ten-fold increase in ion intensity as compared with standard sample preparation methods. The enhanced phosphopeptide response was observed during MALDI-MS analysis of several peptide mixtures derived by proteolytic digestion of phosphoproteins. Furthermore, the mixture of 2,5-DHB and o-phosphoric acid was an excellent eluant for immobilized metal affinity chromatography (IMAC). Singly and multiply phosphorylated peptide species were efficiently recovered from Fe(III)-IMAC columns, reducing sample handling for phosphopeptide mapping by MALDI-MS and subsequent phosphopeptide sequencing by MALDI-MS/MS. The enhanced response of phosphopeptide ions in MALDI facilitates MS/MS of large (>3 kDa) multiply phosphorylated peptide species and reduces the amount of analyte needed for complete characterization of phosphoproteins.     

Dehydrogenation and dehalogenation of amines in MALDI‐TOF MS investigated by isotopic labeling

Secondary and tertiary amines have been reported to form [M–H]⁺ that correspond to dehydrogenation in matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS). In this investigation, we studied the dehydrogenation of amines in MALDI‐TOF MS by isotopic labeling. Aliphatic amines were labeled with deuterium on the methylene of an N‐benzyl group, which resulted in the formation of [M–D]⁺ and [M–H]⁺ ions by dedeuteration and dehydrogenation, respectively. This method revealed the proton that was removed. The spectra of most tertiary amines with an N‐benzyl group showed high‐intensity [M–D]⁺ and [M–H]⁺ ion peaks, whereas those of secondary amines showed low‐intensity ion peaks. Ratios between the peak intensities of [M–D]⁺ and [M–H]⁺ greater than 1 suggested chemoselective dehydrogenation at the N‐benzyl groups. The presence of an electron donor group on the N‐benzyl groups enhanced the selectivity. The dehalogenation of amines with an N‐(4‐halobenzyl) group was also observed alongside dehydrogenation. The amino ions from dehalogenation can undergo second dehydrogenation. These results provide the first direct evidence about the position at which dehydrogenation of an amine occurs and the first example of dehalogenation of haloaromatic compounds in MALDI‐TOF MS. These results should be helpful in the structural identification and elucidation of synthetic and natural molecules. Copyright © 2013 John Wiley & Sons, Ltd.     

Phosphorylation site localization in peptides by MALDI MS/MS and the Mascot Delta Score

Owing to its broad biological significance, the large-scale analysis of protein phosphorylation is more and more getting into the focus of proteomic research. Thousands of phosphopeptides can nowadays be identified using state-of-the-art tandem mass spectrometers in conjunction with sequence database searching, but localizing the phosphate group to a particular amino acid in the peptide sequence is often still difficult. Using 180 individually synthesized phosphopeptides with precisely known phosphorylation sites (p-sites), we have assessed the merits of the Mascot Delta Score (MD score) for the assignment of phosphorylation sites from tandem mass spectra (MS/MS) generated on four different matrix-assisted laser desorption ionization (MALDI) mass spectrometers including tandem time-of-flight (TOF/TOF), quadrupole time-of-flight, and ion trap mass analyzers. The results show that phosphorylation site identification is generally possible with false localization rates of about 10%. However, a comparison to previous work also revealed that phosphorylation site determination by MALDI MS/MS is less accurate than by ESI-MS/MS particularly if several and/or adjacent possible phosphorylation acceptor sites exist in a peptide sequence. We are making the tandem MS spectra and phosphopeptide collection available to the community so that scientists may adapt the MD scores reported here to their analytical environment and so that informatics developers may integrate the MD score into proteomic data analysis pipelines.