Fragmentation behavior of Amadori‐peptides obtained by non‐enzymatic glycosylation of lysine residues with ADP‐ribose in tandem mass spectrometry

Mono‐ and poly‐adenosine diphosphate (ADP)‐ribosylation are common post‐translational modifications incorporated by sequence‐specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non‐enzymatic ADP‐ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori‐compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix‐assisted laser desorption/ionization (MALDI)‐MS, the ADP‐ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in‐source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)‐MS. The same fragmentation site also dominated the MALDI‐PSD (post‐source decay) and ESI‐CID (collision‐induced dissociation) mass spectra. The remaining phospho‐ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b‐ and y‐ion series. Cleavage of the ADP‐ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor‐ion scans (m/z 348.08), and thereby permits the identification of ADP‐ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP‐ribosyl group was stable, providing ADP‐ribosylated c‐ and z‐ions, and thus allowing reliable sequence analyses. Copyright © 2010 John Wiley & Sons, Ltd.     

Applications of mass spectrometry to food proteins and peptides

The application of mass spectrometry (MS) to large biomolecules has been revolutionized in the past decade with the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) techniques. ESI and MALDI permit solvent evaporation and sublimation of large biomolecules into the gaseous phase, respectively. The coupling of ESI or MALDI to an appropriate mass spectrometer has allowed the determination of accurate molecular mass and the detection of chemical modification at high sensitivity (picomole to femtomole). The interface of mass spectrometry hardware with computers and new extended mass spectrometric methods has resulted in the use of MS for protein sequencing, post-translational modifications, protein conformations (native, denatured, folding intermediates), protein folding/unfolding, and protein-protein or protein-ligand interactions. In this review, applications of MS, particularly ESI-MS and MALDI time-of-flight MS, to food proteins and peptides are described.     

Analysis of antioxidants in insulation cladding of copper wire: a comparison of different mass spectrometric techniques (ESI–IT,MALDI–RTOF and RTOF–SIMS)

Commercial copper wire and its polymer insulation cladding was investigated for the presence of three synthetic antioxidants (ADK STAB AO412S, Irganox 1010 and Irganox MD 1024) by three different mass spectrometric techniques including electrospray ionization–ion trap–mass spectrometry (ESI–IT–MS), matrix‐assisted laser desorption/ionization reflectron time‐of‐flight (TOF) mass spectrometry (MALDI–RTOF–MS) and reflectron TOF secondary ion mass spectrometry (RTOF–SIMS). The samples were analyzed either directly without any treatment (RTOF–SIMS) or after a simple liquid/liquid extraction step (ESI–IT–MS, MALDI–RTOF–MS and RTOF–SIMS). Direct analysis of the copper wire itself or of the insulation cladding by RTOF–SIMS allowed the detection of at least two of the three antioxidants but at rather low sensitivity as molecular radical cations and with fairly strong fragmentation (due to the highly energetic ion beam of the primary ion gun). ESI–IT‐ and MALDI–RTOF–MS‐generated abundant protonated and/or cationized molecules (ammoniated or sodiated) from the liquid/liquid extract. Only ESI–IT–MS allowed simultaneous detection of all three analytes in the extract of insulation claddings. The latter two so‐called ‘soft’ desorption/ionization techniques exhibited intense fragmentation only by applying low‐energy collision‐induced dissociation (CID) tandem MS on a multistage ion trap‐instrument and high‐energy CID on a tandem TOF‐instrument (TOF/RTOF), respectively. Strong differences in the fragmentation behavior of the three analytes could be observed between the different CID spectra obtained from either the IT‐instrument (collision energy in the very low eV range) or the TOF/RTOF‐instrument (collision energy 20 keV), but both delivered important structural information. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

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.     

The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.     

Peptides derivatized with bicyclic quaternary ammonium ionization tags. Sequencing via tandem mass spectrometry

Improving the sensitivity of detection and fragmentation of peptides to provide reliable sequencing of peptides is an important goal of mass spectrometric analysis. Peptides derivatized by bicyclic quaternary ammonium ionization tags: 1‐azabicyclo[2.2.2]octane (ABCO) or 1,4‐diazabicyclo[2.2.2]octane (DABCO), are characterized by an increased detection sensitivity in electrospray ionization mass spectrometry (ESI‐MS) and longer retention times on the reverse‐phase (RP) chromatography columns. The improvement of the detection limit was observed even for peptides dissolved in 10 mM NaCl. Collision‐induced dissociation tandem mass spectrometry of quaternary ammonium salts derivatives of peptides showed dominant a‐ and b‐type ions, allowing facile sequencing of peptides. The bicyclic ionization tags are stable in collision‐induced dissociation experiments, and the resulted fragmentation pattern is not significantly influenced by either acidic or basic amino acid residues in the peptide sequence. Obtained results indicate the general usefulness of the bicyclic quaternary ammonium ionization tags for ESI‐MS/MS sequencing of peptides. Copyright © 2014 John Wiley & Sons, Ltd.     

Electrospray ionization and atmospheric pressure matrix‐assisted laser desorption/ionization mass spectrometry of antioxidants applied in lubricants

The aim of this study was to investigate the utility of ion trap mass spectrometry (ITMS) in combination with the two desorption/ionization methods, electrospray (ESI) and atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI), for the detection of antioxidants which are applied in lubricants. These experiments should form the base for future investigations of antioxidants in tribologically formed thin layers on the surface of frictional systems. Seventeen different antioxidants were selected out of the group of hindered phenolic and aromatic aminic compounds. Practically all antioxidants could be characterized by positive ion ESI‐ and AP‐MALDI‐ITMS, forming various types/species of molecular ions (e.g. [M]^{+ .} , [M+H]⁺, [M+Na]⁺ or [M–2H+H]⁺). A few compounds could be analyzed by negative ion ESI‐MS, too, but none by negative ion AP‐MALDI‐MS. The influence of target materials in AP‐MALDI‐MS (gold‐ and titanium nitride (TiN)‐covered stainless steel, micro‐diamond‐covered hard metal, hand‐polished and sand‐blasted stainless steel targets) with respect to the molecular ion intensity and type of molecular ion of two selected antioxidants was evaluated. The surface properties are of particular interest because in friction tests different materials with different surface characteristics are used. However, the MS results indicate that optimal target surfaces have to be found for individual antioxidants in AP‐MALDI‐MS but in general smooth surfaces were superior to rough surfaces. Finally the gold‐covered stainless steel MALDI target provided the best mass spectra and was selected for all the antioxidants investigated. Copyright © 2009 John Wiley & Sons, Ltd.     

Theoretical investigation of low detection sensitivity for underivatized carbohydrates in ESI and MALDI

Electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) mainly generate protonated ions from peptides and proteins but sodiated (or potassiated) ions from carbohydrates. The ion intensities of sodiated (or potassiated) carbohydrates generated by ESI and MALDI are generally lower than those of protonated peptides and proteins. Ab initio calculations and transition state theory were used to investigate the reasons for the low detection sensitivity for underivatized carbohydrates. We used glucose and cellobiose as examples and showed that the low detection sensitivity is partly attributable to the following factors. First, glucose exhibits a low proton affinity. Most protons generated by ESI or MALDI attach to water clusters and matrix molecules. Second, protonated glucose and cellobiose can easily undergo dehydration reactions. Third, the sodiation affinities of glucose and cellobiose are small. Some sodiated glucose and cellobiose dissociate into the sodium cations and neutral carbohydrates during ESI or MALDI process. The increase of detection sensitivity of carbohydrates in mass spectrometry by various methods can be rationalized according to these factors. Copyright © 2016 John Wiley & Sons, Ltd.     

Investigation of tyrosine nitration in proteins by mass spectrometry.

In vivo nitration of tyrosine residues is a post-translational modification mediated by peroxynitrite that may be involved in a number of diseases. The aim of this study was to evaluate possibilities for site-specific detection of tyrosine nitration by mass spectrometry. Angiotensin II and bovine serum albumin (BSA) nitrated with tetranitromethane (TNM) were used as model compounds. Three strategies were investigated: (i) analysis of single peptides and protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping, (ii) peptide mass mapping by electrospray ionization (ESI) mass spectrometry and (iii) screening for nitration by selective detection of the immonium ion of nitrotyrosine by precursor ion scanning with subsequent sequencing of the modified peptides. The MALDI time-of-flight mass spectrum of nitrated angiotensin II showed an unexpected prompt fragmentation involving the nitro group, in contrast to ESI-MS, where no fragmentation of nitrated angiotensin II was observed. The ESI mass spectra showed that mono- and dinitrated angiotensin II were obtained after treatment with TNM. ESI-MS/MS revealed that the mononitrated angiotensin II was nitrated on the side-chain of tyrosine. The dinitrated angiotensin II contained two nitro groups on the tyrosine residue. Nitration of BSA was confirmed by Western blotting with an antibody against nitrotyrosine and the sites for nitration were investigated by peptide mass mapping after in-gel digestion. Direct mass mapping by ESI revealed that two peptides were nitrated. Precursor ion scanning for the immonium ion for nitrotyrosine revealed two additional partially nitrated peptides. Based on the studies with the two model compounds, we suggest that the investigation of in vivo nitration of tyrosine and identification of nitrated peptides might be performed by precursor ion scanning for the specific immonium ion at m/z 181.06 combined with ESI-MS/MS for identification of the specific nitration sites.     

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.     

Fourier transform ion cyclotron resonance mass spectrometry with NanoLC/microelectrospray ionization and matrix-assisted laser desorption/ionization: analytical performance in peptide mass fingerprint analysis

Protein identifications by peptide mass fingerprint analyses with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were performed using microelectrospray ionization coupled to nano liquid chromatography (NanoLC), as well as using matrix-assisted laser desorption/ionization (MALDI). Tryptic digests of bovine serum albumin (BSA), diluted down to femtomole quantities, have been desalted by fast NanoLC under isocratic elution conditions as the high resolving power of FT-ICR MS enables peptides to be separated during the mass analysis stage of the experiment. The high mass accuracy achieved with FT-ICR MS (a few ppm with external calibration) facilitated unambiguous protein identification from protein database searches, even when only a few tryptic peptides of a protein were detected. Statistical confidence in the database search results was further improved by internal calibration due to increased mass accuracy. Matrix-assisted laser desorption/ionization and micro electrospray ionization (ESI) FT-ICR showed good mass accuracies in the low femtomole range, yet a better sensitivity was observed with MALDI. However, in higher femtomole ranges slightly lower mass accuracies were observed with MALDI FT-ICR than with microESI FT-ICR due to scan-to-scan variations of the ion population in the ICR cell. Database search results and protein sequence coverage results from NanoLC FT-ICR MS and MALDI FT-ICR MS, as well as the effect of mass accuracy on protein identification for the peptide mass fingerprint analysis are evaluated.     

Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization by sheathless CZE‐ESI‐MS/MS

Amino acids residues are commonly submitted to various physicochemical modifications occurring at physiological pH and temperature. Post‐translational modifications (PTMs) require comprehensive characterization because of their major influence on protein structure and involvement in numerous in vivo process or signaling. Mass spectrometry (MS) has gradually become an analytical tool of choice to characterize PTMs; however, some modifications are still challenging because of sample faint modification levels or difficulty to separate an intact peptide from modified counterparts before their transfer to the ionization source. Here, we report the implementation of capillary zone electrophoresis coupled to electrospray ionization tandem mass spectrometry (CZE‐ESI‐MS/MS) by the intermediate of a sheathless interfacing for independent and highly sensitive characterization of asparagine deamidation (deaN) and aspartic acid isomerization (isoD). CZE selectivity regarding deaN and isoD was studied extensively using different sets of synthetic peptides based on actual tryptic peptides. Results demonstrated CZE ability to separate the unmodified peptide from modified homologous exhibiting deaN, isoD or both independently with a resolution systematically superior to 1.29. Developed CZE‐ESI‐MS/MS method was applied for the characterization of monoclonal antibodies and complex protein mixture. Conserved CZE selectivity could be demonstrated even for complex samples, and foremost results obtained showed that CZE selectivity is similar regardless of the composition of the peptide. Separation of modified peptides prior to the MS analysis allowed to characterize and estimate modification levels of the sample independently for deaN and isoD even for peptides affected by both modifications and, as a consequence, enables to distinguish the formation of l ‐aspartic acid or d ‐aspartic acid generated from deaN. Separation based on peptide modification allowed, as supported by the ESI efficiency provided by CZE‐ESI‐MS/MS properties, and enabled to characterize and estimate studied PTMs with an unprecedented sensitivity and proved the relevance of implementing an electrophoretic driven separation for MS‐based peptide analysis. Copyright © 2016 John Wiley & Sons, Ltd.     

Protein identification by accurate mass matrix-assisted laser desorption/ionization imaging of tryptic peptides

The spatial distribution of proteins in tissue sections can be used to identify potential markers for pathological processes. Tissue sections are often subjected to enzymatic digestion before matrix‐assisted laser desorption/ionization (MALDI) imaging. This study is targeted at improving the on‐tissue identification of tryptic peptides by accurate mass measurements and complementary off‐line liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) analysis. Two adjacent mouse brain sections were analyzed in parallel. The first section was spotted with trypsin and analyzed by MALDI imaging. Direct on‐tissue MS/MS experiments of this section resulted in the identification of 14 peptides (originating from 4 proteins). The second tissue section was homogenized, fractionated by ultracentrifugation and digested with trypsin prior to LC/ESI‐MS/MS analysis. The number of identified peptides was increased to 153 (corresponding to 106 proteins) by matching imaged mass peaks to peptides which were identified in these LC/ESI‐MS/MS experiments. All results (including MALDI imaging data) were based on accurate mass measurements (RMS <2 ppm) and allow a confident identification of tryptic peptides. Measurements based on lower accuracy would have led to ambiguous or misleading results. MS images of identified peptides were generated with a bin width (mass range used for image generation) of Δm/z = 0.01. The application of accurate mass measurements and additional LC/MS measurements increased both the quality and the number of peptide identifications. The advantages of this approach for the analysis of biological tissue sections are demonstrated and discussed in detail. Results indicate that accurate mass measurements are needed for confident identification and specific image generation of tryptic peptides in tissue sections. Copyright © 2011 John Wiley & Sons, Ltd.     

Negative ion production from peptides and proteins by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Negative ion production from peptides and proteins was investigated by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Although most research on peptide and protein identification with ionization by MALDI has involved the detection of positive ions, for some acidic peptides protonated molecules are not easily formed because the side chains of acidic residues are more likely to lose a proton and form a deprotonated species. After investigating more than 30 peptides and proteins in both positive and negative ion modes, [M–H]⁻ ions were detected in the negative ion mode for all peptides and proteins although the matrix used was 2,5‐dihydroxybenzoic acid (DHB), which is a good proton donor and favors the positive ion mode production of [M+H]⁺ ions. Even for highly basic peptides without an acidic site, such as myosin kinase inhibiting peptide and substance P, good negative ion signals were observed. Conversely, gastrin I (1‐14), a peptide without a highly basic site, will form positive ions. In addition, spectra obtained in the negative ion mode are usually cleaner due to absence of alkali metal adducts. This can be useful during precursor ion isolation for MS/MS studies. Copyright © 2008 John Wiley & Sons, Ltd.     

A method to determine the ionization efficiency change of peptides caused by phosphorylation

Quantitative assessment of post-translational modifications in proteins by mass spectrometry often requires the consideration of the alteration in ionization efficiency of peptides induced by the modification. Herein, we introduced a method to measure the relative ionization efficiencies of peptides using specifically designed unlabeled peptides. In our design, the peptide under study, in either the unmodified or modified form, is linked with an internal standard peptide via an enzyme cleavage site; thus, after enzymatic digestion, we could obtain readily a 1:1 ratio between the peptide under investigation and the internal standard peptide. The relative ionization efficiencies of the modified and unmodified peptides can then be calculated from the modification-induced change in the ratio of relative abundances of the ion of the peptide of interest over that of the internal standard peptide. We demonstrated the usefulness of the method by assessing the change in ionization efficiencies of four peptides introduced by phosphorylation.     

Toward milk speciation through the monitoring of casein proteotypic peptides

The possibility of detecting extraneous milk in singles species cheese‐milk has been explored. A mass spectrometry (MS)‐based procedure has been developed to detect 'signature peptides', corresponding to the predefined subset of 'proteotypic peptides', as matchless analytical surrogates of the parent caseins. Tryptic digests of skimmed milk samples from four species were analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS. Amongst the candidate signature peptides that are able to differentiate milks from the four species, the α_s1‐casein (CN) f8‐22 peptide was selected as a convenient marker for bovine, ovine and water buffalo milk while the f4‐22 peptide was selected as a marker for the two caprine α_s1‐CN A and B variants, which differ by a Pro¹⁶ (B)‐>Leu¹⁶ (A) substitution. MALDI analysis of the digest allowed the detection of α_s1‐CN f8‐22 and caprine α_s1‐CN f4‐22. The accurate evaluation of caprine milk in a quaternary mixture required the development of a liquid chromatography/electrospray ionization (LC/ESI)‐MS procedure. Five synthetic signature peptide analogues, which differed from their natural counterparts by a single amino acid substitution, were used as internal standards to quantify the α_s1‐CN, which was chosen as a reference milk protein, from the different species. The limits of detection were 0.5% (1% for caprine) for either the MALDI or the LC/ESI‐MS method. The isotopic‐label‐free quantification of isoform‐ or variant‐specific signature peptides has disclosed a convenient approach for targeting proteins in complex mixtures. Copyright © 2010 John Wiley & Sons, Ltd.     

Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests

Proteomics requires an optimized level of sample-processing, including a minimal sample-processing time and an optimal peptide recovery from protein digests, in order to maximize the percentage sequence coverage and to improve the accuracy of protein identification. The conventional methods of protein characterization from one-dimensional or two-dimensional gels include the destaining of an excised gel piece, followed by an overnight in-gel enzyme digestion. The aims of this study were to determine whether: (1) stained gels can be used without any destaining for trypsin digestion and mass spectrometry (MS); (2) tryptic peptides can be recovered from a matrix-assisted laser desorption/ionization (MALDI) target plate for a subsequent analysis with liquid chromatography (LC) coupled to an electrospray ionization (ESI) quadrupole ion trap MS; and (3) an overnight in-gel digestion is necessary for protein characterization with MS. These three strategies would significantly improve sample throughput. Cerebrospinal fluid (CSF) was the model biological fluid used to develop these methods. CSF was desalted by gel filtration, and CSF proteins were separated by two-dimensional gel electrophoresis (2DGE). Proteins were visualized with either silver, Coomassie, or Stains-All (counterstained with silver). None of the gels was destained. Protein spots were in-gel trypsin digested, the tryptic peptides were purified with ZipTip, and the peptides were analyzed with MALDI and ESI MS. Some of the samples that were spotted onto a wax-coated MALDI target plate were recovered and analyzed with ESI MS. All three types of stained gels were compatible with MALDI and ESI MS without any destaining. In-gel trypsin digestion can be performed in only 10-60 min for protein characterization with MS, the sample can be recovered from the MALDI target plate for use in ESI MS, and there was a 90% reduction in sample-processing time from overnight to ca. 3 h.     

Identification of nuclear proteins of small cell lung cancer cell line H82: An improved procedure for the analysis of silver-stained proteins

An efficient method for digestion and extraction of proteolytic peptides from silver-stained proteins was applied to the characterization of nuclear proteins from the small cell lung cancer H82 (ATCC HTB 175) cell line previously separated by high-resolution large format two-dimensional gel electrophoresis. From 68 spots, evenly distributed on the gel area and representing a wide range of spot intensities, 63 (92%) were successfully identified by matrix-assisted laser desorption/ionization (MALDI) or electrospray ionozation-mass spectrometry (ESI-MS). In five cases where the identification was not possible, the presence of an intense background apparently due to the leakage of polymers from the microtubes or other plastics, was detected. Extensive analysis of peptide sequences by ESI MS/MS experiments allowed the identification of post-translational modifications, such as acetylation, phosphorylation, deamidation of asparagine residues and the presence of isoaspartic acid. A new protein variant not reported in sequence databases was also detected.     

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.