Neutral loss of isocyanic acid in peptide CID spectra: A novel diagnostic marker for mass spectrometric identification of protein citrullination

Protein citrullination is emerging as an important signaling mechanism that modulates a variety of biological processes. This protein modification constitutes only a 1 Da mass shift, and can be readily confused with other common protein modifications that yield an identical mass shift. In an attempt to develop a robust methodology for detection of protein citrullination sites, we analyzed synthetic citrulline-containing peptides by electrospray ionization tandem mass spectrometry. Collision-induced dissociation (CID) spectra revealed abundant neutral loss of 43 Da from citrullinated peptide precursor ions, which was reconciled by elimination of the HNCO moiety (isocyanic acid) from the citrulline ureido group. The elimination occurs readily in multiple charge states of precursor ions and also in b and y ions. HNCO loss in CID spectra provides a novel diagnostic marker for citrullination, and its utility was demonstrated by the discovery of Arg197 as the specific site of citrullination on nucleophosmin upon peptidylarginine deiminase 4 treatment.     

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.     

An integrated serum proteomic approach capable of monitoring the low molecular weight proteome with sequencing of intermediate to large peptides

The low‐abundance, low molecular weight serum proteome has high potential for the discovery of new biomarkers using mass spectrometry (MS). Because the serum proteome is large and complex, defining relative quantitative differences for a molecular species between comparison groups requires an approach with robust separation capability, high sensitivity, as well as high mass resolution. Capillary liquid chromatography (cLC)/MS provides both the necessary separation technique and the sensitivity to observe many low‐abundance peptides. Subsequent identification of potential serum peptide biomarkers observed in the cLC/MS step can in principle be accomplished by in series cLC/MS/MS without further sample preparation or additional instrumentation. In this report a novel cLC/MS/MS method for peptide sequencing is described that surpasses previously reported size limits for amino acid sequencing accomplished by collisional fragmentation using a tandem time‐of‐flight MS instrument. As a demonstration of the approach, two low‐abundance peptides with masses of ～4000–5000 Da were selected for MS/MS sequencing. The multi‐channel analyzer (MCA) was used in a novel way that allowed for summation of 120 fragmentation spectra for each of several customized collision energies, providing more thorough fragmentation coverage of each peptide with improved signal to noise. The peak list from this composite analysis was submitted to Mascot for identification. The two index peptides, 4279 Da and 5061 Da, were successfully identified. The peptides were a 39 amino acid immunoglobulin G heavy chain variable region fragment and a 47 amino acid fibrin alpha isoform C‐terminal fragment. The method described here provides the ability both to survey thousands of serum molecules and to couple that with markedly enhanced cLC/MS/MS peptide sequencing capabilities, providing a promising technique for serum biomarker discovery. Copyright © 2009 John Wiley & Sons, Ltd.     

ESIprot: a universal tool for charge state determination and molecular weight calculation of proteins from electrospray ionization mass spectrometry data

Electrospray ionization (ESI) ion trap mass spectrometers with relatively low resolution are frequently used for the analysis of natural products and peptides. Although ESI spectra of multiply charged protein molecules also can be measured on this type of devices, only average spectra are produced for the majority of naturally occurring proteins. Evaluating such ESI protein spectra would provide valuable information about the native state of investigated proteins. However, no suitable and freely available software could be found which allows the charge state determination and molecular weight calculation of single proteins from average ESI‐MS data. Therefore, an algorithm based on standard deviation optimization (scatter minimization) was implemented for the analysis of protein ESI‐MS data. The resulting software ESIprot was tested with ESI‐MS data of six intact reference proteins between 12.4 and 66.7 kDa. In all cases, the correct charge states could be determined. The obtained absolute mass errors were in a range between ?0.2 and 1.2 Da, the relative errors below 30 ppm. The possible mass accuracy allows for valid conclusions about the actual condition of proteins. Moreover, the ESIprot algorithm demonstrates an extraordinary robustness and allows spectral interpretation from as little as two peaks, given sufficient quality of the provided m/z data, without the necessity for peak intensity data. ESIprot is independent from the raw data format and the computer platform, making it a versatile tool for mass spectrometrists. The program code was released under the open‐source GPLv3 license to support future developments of mass spectrometry software. Copyright © 2010 John Wiley & Sons, Ltd.     

A computer program (COMPOST) for predicting mass spectrometric information from known amino acid sequences

A computer program (COMPOST) is described that carries out predictive computations on known amino acid sequences. The program is designed to be of use to mass spectrometrists with an interest in protein and peptide sequencing. Mass values (monoisotopic and average) for protonated peptide and protein molecules and elemental compositions are calculated. COMPOST also calculates mass to charge ratio values for protonated peptides expected from specified digests, locates specified amino acid subsequences or peptides of a specifIed molecular weight within a longer sequence, and predicts mass to charge ratio values for fragment ions from high-energy collision-induced dissociation of protonated peptides.     

ACE-Inhibitory and Antioxidant Activities of Peptide Fragments Obtained from Tomato Processing By-Products Fermented Using <Emphasis Type="Italic">Bacillus subtilis</Emphasis>: Effect of Amino Acid Composition and Peptides Molecular Mass Distribution

The effects of amino acid composition and peptide molecular mass on ACE-inhibitory and antioxidant activities of protein fragments obtained from tomato waste fermented using Bacillus subtilis were evaluated. The addition of B. subtilis increased the relative amounts of aromatic and positively-charged amino acids which have been described to influence the biological activities of peptide fragments. IC₅₀ values of hydrolysates for ACE-inhibitory and 2, 2′-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities were found to be 1.5 and 8.2 mg/mL, respectively. Size-exclusion chromatography (SEC) pattern of the hydrolysate indicated the breakdown of parent proteins to smaller peptides with molecular weights mainly below 1400 Da. MALDI-TOF mass spectrometry analysis revealed that the highest ACE-inhibitory activity was due to peptides showing molecular mass range 500–800 Da, while the most active antioxidant peptides were found to be mainly at the two different peptide weight ranges 500–800 Da and 1200–1500 Da.     

Effects of peptide acetylation and dimethylation on electrospray ionization efficiency

Peptide acetylation and dimethylation have been widely used to derivatize primary amino groups (peptide N‐termini and the ε‐amino group of lysines) for chemical isotope labeling of quantitative proteomics or for affinity tag labeling for selection and enrichment of labeled peptides. However, peptide acetylation results in signal suppression during electrospray ionization (ESI) due to charge neutralization. In contrast, dimethylated peptides show increased ionization efficiency after derivatization, since dimethylation increases hydrophobicity and maintains a positive charge on the peptide under common LC conditions. In this study, we quantitatively compared the ESI efficiencies of acetylated and dimethylated model peptides and tryptic peptides of BSA. Dimethylated peptides showed higher ionization efficiency than acetylated peptides for both model peptides and tryptic BSA peptides. At the proteome level, peptide dimethylation led to better protein identification than peptide acetylation when tryptic peptides of mouse brain lysate were analyzed with LC‐ESI‐MS/MS. These results demonstrate that dimethylation of tryptic peptides enhanced ESI efficiency and provided up to two‐fold improved protein identification sensitivity in comparison with acetylation. Copyright © 2016 John Wiley & Sons, Ltd.     

Modification of citrulline residues with 2,3-butanedione facilitates their detection by liquid chromatography/mass spectrometry

Citrullination is a post-translational modification (PTM) that results from the deimination of the amino acid arginine into citrulline by Peptidyl Arginine Deiminase enzymes and occurs in a wide range of proteins in health and disease. This modification causes a 1 Da mass shift, which can be used to identify citrullination sites in proteins by the use of mass spectrometry. However, other PTMs, such as deamidation from asparagine to aspartic acid or from glutamine to glutamic acid, can also cause a 1 Da mass shift, making correct interpretation of the data more difficult. We developed a chemical tagging strategy which, combined with an open source search application, allowed us to selectively pinpoint citrullinated peptides in a complex mixture after liquid chromatography/mass spectrometry (LC/MS) analysis. After incubation of a peptide mixture with 2,3 butanedione, citrulline residues were covalently modified which resulted in a 50 Da shift in singly charged mass. By comparison of the peptide mass fingerprint from a modified and an unmodified version of the same sample, our in-house search application was able to identify the citrullinated peptides in the mixture. This strategy was optimized on synthetic peptides and validated on a digest of in vitro citrullinated fibrinogen, where different proteolytic enzymes were used to augment the protein coverage. This new method results in easy detection of citrullinated residues, without the need for complex mass spectrometry equipment.     

A hevein-like protein and a class I chitinase with antifungal activity from leaves of the paper mulberry

Paper mulberry (Broussonetia papyrifera, syn. Morus papyrifera L.) is a Chinese traditional medicine and its low‐molecular‐weight extracts are reported to have antifungal activity. In this study, two proteins (PMAPI and PMAPII) with activity against Trichoderma viride were obtained from paper mulberry leaves with a fast protein liquid chromatography (FPLC) unit. The purification protocol employed (NH₄)₂SO₄ precipitation, ion‐exchange chromatography and hydrophobic‐interaction chromatography on FPLC. Molecular masses were 18,798 Da for PMAPI, and 31,178 Da for PMAPII determined by Matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Peptide mapping fingerprint analysis showed that PMAPI has no peptides similar to PMAPII. N‐terminal amino acid sequencing revealed that PMAPI is a hevein‐like protein, and PMAPII is a class I chitinase. They both had a half‐maximal inhibitory concentration (IC50) of 0.1 µg/µL against T. viride. This is the first report of high‐molecular‐weight extracts with antifungal activity from paper mulberry. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

A fast,reproducible and low‐cost method for sequence deconvolution of ‘on‐bead’ peptides via ‘on‐target’ maldi‐TOF/TOF mass spectrometry

A novel approach to high‐throughput sequence deconvolution of on‐bead small peptides (MW < 2000 Da) using on‐target MALDI‐TOF/TOF instrumentation is presented. Short peptides of pentamer and octamer length, covalently attached to TentaGel polystyrene beads through a photolabile linker, were placed onto the MALDI target, apportioned with suitable matrix (2,5‐dihydroxybenzoic acid) and then hit with the instrument laser (Nd : YAG, 355 nm). This induced easy and highly reproducible photochemical cleavage, desorption (MS mode) and fragmentation (MS/MS mode). Peptide fragments were identified with a mass accuracy of 0.1 Da of the expected values. This technique significantly accelerates the sequence determination of positive peptide hits obtained from random combinatorial libraries when screening against biological targets, paving the way for a rapid and efficient method to identify molecular imaging ligands specific to pathological targets in cancer and other diseases. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of unique amino acid substitutions in protein variants by peptide mass mapping with FT-ICR MS

Peptide mass mapping plays a central role in the structural characterization of protein variants with single amino acid substitutions. Among the 20 standard amino acids found in living organisms, 18, all but Leu and Ile, differ from each other in molecular mass. The mass differences between amino acids range from 0.0364 to 129.0578 Da. The mass of the mutated peptide or the difference between normal and mutated peptides uniquely determines the type of substitution in some cases, and even pinpoints the position of the mutation when the involved residue is found only once in the peptide. Among 75 pairs of amino acid residues that are exchangeable via a single nucleotide replacement, 53 show specific change in exact mass, while only 25 in nominal mass. On the other hand, precise measurement, at least to the third decimal place, greatly enhances the capacity of the peptide mass mapping strategy for structural characterization. This notion was verified by an analysis of three Hb variants using MALDI-FTICR MS. In addition, the baseline resolution of two 1 kDa peptides with a single amino acid difference, Lys or Gln, which have the smallest (0.0364 Da) difference among residues, was achieved by measurement at a mass resolving power of 342,000. The results indicated that the smallest difference, 0.0040 Da between [Delta29.9742 for Glu-Val] and [Delta29.9782 for Trp-Arg], among all types of amino acid substitutions derived from a single nucleotide replacement can be discriminated at the present performance level. Therefore, FTICR MS is capable of identifying all 53 types of substitutions, each of which is associated with a unique mass difference, except for the Leu and Ile isomers.     

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。     

A Novel Triethylphosphonium Charge Tag on Peptides: Synthesis,Derivatization, and Fragmentation

Charge tagging is a peptide derivatization process that commonly localizes a positive charge on the N-terminus. Upon low energy activation (e.g., collision-induced dissociation or post-source decay) of charge tagged peptides, relatively few fragment ions are produced due to the absence of mobile protons. In contrast, high energy fragmentation, such as 157 nm photodissociation, typically leads to a series of a-type ions. Disadvantages of existing charge tags are that they can produce mobile protons or that they are undesirably large and bulky. Here, we investigate a small triethylphosphonium charge tag with two different linkages: amide (158 Da) and amidine bonds (157 Da). Activation of peptides labeled with a triethylphosphonium charge tag through an amide bond can lead to loss of the charge tag and the production of protonated peptides. This enables low intensity fragment ions from both the protonated and charge tagged peptides to be observed. Triethylphosphonium charge tagged peptides linked through an amidine bond are more stable. Post-source decay and photodissociation yield product ions that primarily contain the charge tag. Certain amidine induced fragments are also observed. The previously reported tris(trimethoxyphenyl) phosphonium acetic acid N-hydroxysuccinimidyl ester charge tag shows a similar fragment ion distribution, but the mass of the triethylphosphonium tag label is 415 Da smaller.

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Determination of pyridoxal‐5′‐phosphate (PLP)‐bonding sites in proteins: a peptide mass fingerprinting approach based on diagnostic tandem mass spectral features of PLP‐modified peptides

Peptides modified by pyridoxal‐5′‐phosphate (PLP), linked to a lysine residue via reductive amination, exhibit distinct spectral characteristics in the collision‐induced dissociation (CID) tandem mass (MS/MS) spectra that are described here. The MS/MS spectra typically display two dominant peaks whose m/z values correspond to neutral losses of [H₃PO₄] (?98 Da) and the PLP moiety as [C₈H₁₀NO₅P] (?231 Da) from the precursor peptide ion, respectively. Few other peaks are observed. Recognition of this distinct fragmentation behavior is imperative since determining sequences and sites of modifications relies on the formation of amide backbone cleavage products for subsequent interpretation via proteome database searching. Additionally, PLP‐modified peptides exhibit suppressed precursor ionization efficiency which diminishes their detection in complex mixtures. Presented here is a protocol which describes an enrichment strategy for PLP‐modified peptides combined with neutral loss screening and peptide mass fingerprinting to map the PLP‐bonding site in a known PLP‐dependent protein. This approach represents an efficient alternative to site‐directed mutagenesis which has been the traditional method used for PLP‐bonding site localization in proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Proteomic analysis of peptides tagged with dimedone and related probes

Owing to its labile nature, a new role for cysteine sulfenic acid (–SOH) modification has emerged. This oxidative modification modulates protein function by acting as a redox switch during cellular signaling. The identification of proteins that undergo this modification represents a methodological challenge, and its resolution remains a matter of current interest. The development of strategies to chemically modify cysteinyl‐containing peptides for liquid chromatography–tandem mass spectrometry (LC‐MS/MS) analysis has increased significantly within the past decade. The method of choice to selectively label sulfenic acid is based on the use of dimedone or its derivatives. For these chemical probes to be effective on a proteome‐wide level, their reactivity toward –SOH must be high to ensure reaction completion. In addition, the presence of an adduct should not interfere with electrospray ionization, the efficiency of induced dissociation in MS/MS experiments or with the identification of Cys‐modified peptides by automated database searching algorithms. Herein, we employ a targeted proteomics approach to study the electrospray ionization and fragmentation effects of different –SOH specific probes and compared them to commonly used alkylating agents. We then extend our study to a whole proteome extract using shotgun proteomic approaches. These experiments enable us to demonstrate that dimedone adducts do not interfere with electrospray by suppressing the ionization nor impede product ion assignment by automated search engines, which detect a + 138 Da increase from unmodified peptides. Collectively, these results suggest that dimedone can be a powerful tool to identify sulfenic acid modifications by high‐throughput shotgun proteomics of a whole proteome. Copyright © 2014 John Wiley & Sons, Ltd.     

Mass spectrometric identification of formaldehyde-induced peptide modifications under in vivo protein cross-linking conditions

Formaldehyde cross-linking of proteins is emerging as a novel approach to study protein-protein interactions in living cells. It has been shown to be compatible with standard techniques used in functional proteomics such as affinity-based protein enrichment, enzymatic digestion, and mass spectrometric protein identification. So far, the lack of knowledge on formaldehyde-induced protein modifications and suitable mass spectrometric methods for their targeted detection has impeded the identification of the different types of cross-linked peptides in these samples. In particular, it has remained unclear whether in vitro studies that identified a multitude of amino acid residues reacting with formaldehyde over the course of several days are suitable substitutes for the much shorter reaction times of 10-20 min used in cross-linking experiments in living cells. The current study on model peptides identifies amino-termini as well as lysine, tryptophan, and cysteine side chains, i.e. a small subset of those modified after several days, as the major reactive sites under such conditions, and suggests relative position in the peptide sequence as well as sequence microenvironment to be important factors that govern reactivity. Using MALDI-MS, mass increases of 12 Da on amino groups and 30 Da on cysteines were detected as the major reaction products, while peptide fragment ion analysis by tandem mass spectrometry was used to localize the actual modification sites on a peptide. Non-specific cross-linking was absent, and could only be detected with low yield at elevated peptide concentrations. The detailed knowledge on the constraints and products of the formaldehyde reaction with peptides after short incubation times presented in this study is expected to facilitate the targeted mass spectrometric analysis of proteins after in vivo formaldehyde cross-linking.     

Identification and characterization of cysteinyl exposure in proteins by selective mercury labeling and nano‐electrospray ionization quadrupole time‐of‐flight mass spectrometry

We describe a method for probing surface‐exposed cysteines in proteins by selective labeling with p‐hydroxymercuribenzoate (PMB) combined with nano‐electrospray ionization mass spectrometric analysis (nanoESI‐MS). The rapid, stoichiometric, and specific labeling by PMB of surface‐exposed cysteines allows for characterization of the accessibility of the cysteines using a single MS analysis. Moreover, by taking advantage of the large mass shift of 321 Da, unique isotopic pattern, and enhanced MS signal of PMB‐labeled cysteine‐containing peptide fragments, the surface‐exposed cysteines in proteins can be accurately identified by peptide mapping. The number and sites of reactive cysteines on the surface of human and rat hemoglobins (hHb and rHb) were identified as examples. Collision‐induced dissociation tandem mass spectrometric (MS/MS) analysis of specific peptides further confirmed the selective labeling of PMB in hHb. The subtle difference between the different cysteine residues in rHb was also evaluated by multiple PMB titrations. The difference between the two cysteines in their environment may partially explain their reaction specificity. Cysteine 125 in the β unit of rHb is exposed on the surface, explaining its reactivity with glutathione. Cysteine 13 in the α subunit of rHb is much less exposed, and is located in a hydrophobic pocket, a conclusion that is consistent with the previous observation of its selective binding with dimethylarsinous acid, a reactive arsenic metabolite. The method is potentially useful for probing cysteines in other biologically important proteins and for studying proteins that are associated with conformational or structural changes induced by denaturing processes, protein modifications, protein‐protein interactions and protein assemblies. Copyright © 2010 John Wiley & Sons, Ltd.     

Improved spectra for MALDI MSI of peptides using ammonium phosphate monobasic in MALDI matrix

MALDI mass spectrometry imaging (MSI) enables analysis of peptides along with histology. However, there are several critical steps in MALDI MSI of peptides, 1 of which is spectral quality. Suppression of MALDI matrix clusters by the aid of ammonium salts in MALDI experiments is well known. It is asserted that addition of ammonium salts dissociates potential matrix adducts and thereafter decreases matrix cluster formation. Consequently, MALDI MS sensitivity and mass accuracy increase. Up to our knowledge, a limited number of MALDI MSI studies used ammonium salts as matrix additives to suppress matrix clusters and enhance peptide signals. In this work, we investigated the effect of ammonium phosphate monobasic (AmP) as alpha‐cyano‐4‐hydroxycinnamic acid (α‐CHCA) matrix additive in MALDI MSI of peptides. Prior to MALDI MSI, the effect of varying concentrations of AmP in α‐CHCA was assessed in bovine serum albumin tryptic digests and compared with the control (α‐CHCA without AmP). Based on our data, the addition of AmP as matrix additive decreased matrix cluster formation regardless of its concentration, and specifically, 8 mM AmP and 10 mM AmP increased bovine serum albumin peptide signal intensities. In MALDI MSI of peptides, both 8 and 10 mM AmP in α‐CHCA improved peptide signals especially in the mass range of m/z 2000 to 3000. In particular, 9 peptide signals were found to have differential intensities within the tissues deposited with AmP in α‐CHCA (AUC > 0.60). To the best of our knowledge, this is the first MALDI MSI of peptides work investigating different concentrations of AmP as α‐CHCA matrix additive to enhance peptide signals in formalin‐fixed paraffin‐embedded (FFPE) tissues. Further, AmP as part of α‐CHCA matrix could enhance protein identifications and support MALDI MSI‐based proteomic approaches.     

Characterization of dehydroascorbate‐mediated modification of glutaredoxin by mass spectrometry

Ascorbate is as a potent antioxidant in vivo protecting the organism against oxidative stress. In this process, ascorbate is oxidized in two steps to dehydroascorbate (DHA), which if not efficiently reduced back to ascorbate decomposes irreversibly to a complex mixture of products. We demonstrate that a component of this mixture specifically reacts with the thiol group of cysteine residues at physiological pH to give a protein adduct involving the addition of a 5‐carbon fragment of DHA (+112 Da). Incubations of glutaredoxin‐1 expressed in Escherichia coli and dehydroascorbate revealed abundant adducts of +112, +224 and +336 Da due to the addition of one, two and three conjugation products of DHA, respectively. ESI–MS of carbamidomethylated glutaredoxin‐1 before incubation with DHA, deuterium exchange together with tandem mass spectrometry analysis and LC–ESIMS/MS of modified peptides confirmed structure and sites of modification in the protein. Modification of protein thiols by a DHA‐derived product can be involved in oxidative stress‐mediated cellular toxicity. Copyright © 2015 John Wiley & Sons, Ltd.