Identification of neuropeptides in rat brain rhinencephalon

A capillary two-dimensional liquid chromatography method coupled with ion trap mass spectrometry has been used for separation and identification of neuropeptides in rat rhinencephalon. Animals of three different age groups were exposed to slow and quick CO2 influx. The neuropeptides were extracted by solid phase extraction and the purified extracts were analysed by 2-D HPLC. The compounds were fractionated (strong cation exchange column), trapped and separated, and MS/MS fragment mass spectra were used for identification. About thirty peptide compounds were identified. A significant difference between concentration levels of "stressed" (quick CO2 influx) and "non-stressed" (slow CO2 influx) rats was found for 25 of the identified peptides.     

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.     

Identification of peptides in antimicrobial fractions of cheese extracts by electrospray ionization ion trap mass spectrometry coupled to a two-dimensional liquid chromatographic separation

Electrospray ionization ion trap mass spectrometry (ESI-ITMS) coupled to a two-dimensional liquid chromatographic separation was applied to the identification of peptides in antimicrobial fractions of the aqueous extracts of nine Italian cheese varieties. In particular, the chromatographic fractions collected during a preliminary fast protein liquid chromatography (FPLC) separation on the cheese extracts were assayed for antimicrobial activity towards Lactobacillus sakei A15. Active fractions were subsequently analyzed by reversed-phase high-performance liquid chromatography electrospray ionization sequential mass spectrometry (HPLC/ESI)-ITMSn, with n up to 3. Peptide identification was then performed starting from a conventional proteomics approach based on tandem mass spectrometric (MS/MS) analysis followed by database searching. In many cases this strategy had to be integrated by a careful correlation between spectral information and predicted peptide fragmentation, in order to reach unambiguous identifications. When even this integrated approach failed, MS3 measurements provided decisive information on the amino acid sequence of some peptides, through fragmentation of pendant groups along the peptide chain. As a result, 45 peptides, all arising from hydrolysis of milk caseins, were identified in nine antimicrobial FPLC fractions of aqueous extracts obtained from five of the nine cheese varieties considered. Many of them corresponded to peptides already known to exhibit biological activity.     

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.     

Improving peptide identification using an empirical peptide retention time database

Peptide retention time (RT) is independent of tandem mass spectrometry (MS/MS) parameters and can be combined with MS/MS information to enhance peptide identification. In this paper, we utilized peptide empirical RT and MS/MS for peptide identification. This new approach resulted in the construction of an Empirical Peptide Retention Time Database (EPRTD) based on peptides showing a false‐positive rate (FPR) ≤1%, detected in several liquid chromatography (LC)/MS/MS analyses. In subsequent experiments, the RT of peptides with FPR >1% was compared with empirical data derived from the EPRTD. If the experimental RT was within a specified time range of the empirical value, the corresponding MS/MS spectra were accepted as positive. Application of the EPRTD approach to simple samples (known protein mixtures) and complex samples (human urinary proteome) revealed that this method could significantly enhance peptide identification without compromising the associated confidence levels. Further analysis indicated that the EPRTD approach could improve low‐abundance peptides and with the expansion of the EPRTD the number of peptide identifications will be increased. This approach is suitable for large‐scale clinical proteomics research, in which tens of LC/MS/MS analyses are run for different samples with similar components. Copyright © 2008 John Wiley & Sons, Ltd.     

Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification

Simple and efficient digestion of proteins, particularly hydrophobic membrane proteins, is of significance for comprehensive proteome analysis using the bottom-up approach. We report a microwave-assisted acid hydrolysis (MAAH) method for rapid protein degradation for peptide mass mapping and tandem mass spectrometric analysis of peptides for protein identification. It uses 25% trifluoroacetic acid (TFA) aqueous solution to dissolve or suspend proteins, followed by microwave irradiation for 10 min. This detergent-free method generates peptide mixtures that can be directly analyzed by liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) without the need of extensive sample cleanup. LC-MALDI MS/MS analysis of the hydrolysate from 5 microg of a model transmembrane protein, bacteriorhodopsin, resulted in almost complete sequence coverage by the peptides detected, including the identification of two posttranslational modification sites. Cleavage of peptide bonds inside all seven transmembrane domains took place, generating peptides of sizes amenable to MS/MS to determine possible sequence errors or modifications within these domains. Cleavage specificity, such as glycine residue cleavage, was observed. Terminal peptides were found to be present in relatively high abundance in the hydrolysate, particularly when low concentrations of proteins were used for MAAH. It was shown that these peptides could still be detected from MAAH of bacteriorhodopsin at a protein concentration of 1 ng/microl or 37 fmol/microl. To evaluate the general applicability of this method, it was applied to identify proteins from a membrane protein enriched fraction of cell lysates of human breast cancer cell line MCF7. With one-dimensional LC-MALDI MS/MS, a total of 119 proteins, including 41 membrane-associated or membrane proteins containing one to 12 transmembrane domains, were identified by MS/MS database searching based on matches of at least two peptides to a protein.     

Reproducible microwave-assisted acid hydrolysis of proteins using a household microwave oven and its combination with LC-ESI MS/MS for mapping protein sequences and modifications

A new set-up for microwave-assisted acid hydrolysis (MAAH) with high efficiency and reproducibility to degrade proteins into peptides for mass spectrometry analysis is described. It is based on the use of an inexpensive domestic microwave oven and can be used for low volume protein solution digestion. This set-up has been combined with liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI QTOF MS) for mapping protein sequences and characterizing phosphoproteins. It is demonstrated that for bovine serum albumin (BSA), with a molecular mass of about 67,000 Da, 1292 peptides (669 unique sequences) can be detected from a 2 μg hydrolysate generated by trifluoroacetic acid (TFA) MAAH. These peptides cover the entire protein sequence, allowing the identification of an amino acid substitution in a natural variant of BSA. It is shown that for a simple phosphoprotein containing one phosphoform, β-casein, direct analysis of the hydrolysate generates a comprehensive peptide map that can be used to identify all five known phosphorylation sites. For characterizing a complex phosphoprotein consisting of different phosphoforms with varying numbers of phosphate groups and/or phosphorylation sites, such as bovine α_s1-casein, immobilized metal-ion affinity chromatography (IMAC) is used to enrich the phosphopeptides from the hydrolysate, followed by LC-ESI MS analysis. The MS/MS data generated from the initial hydrolysate and the phosphopeptide-enriched fraction, in combination with MS analysis of the intact protein sample, allow us to reveal the presence of three different phosphoforms of bovine α_s1-casein and assign the phosphorylation sites to each phosphoform with high confidence.     

MSQ: a tool for quantification of proteomics data generated by a liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry based targeted quantitative proteomics platform

Mass spectrometry (MS)‐based quantitative proteomics has become a critical component of biological and clinical research for identification of biomarkers that can be used for early detection of diseases. In particular, MS‐based targeted quantitative proteomics has been recently developed for the detection and validation of biomarker candidates in complex biological samples. In such approaches, synthetic reference peptides that are the stable isotope labeled version of proteotypic peptides of proteins to be quantitated are used as internal standards enabling specific identification and absolute quantification of targeted peptides. The quantification of targeted peptides is achieved using the intensity ratio of a native peptide to the corresponding reference peptide whose spike‐in amount is known. However, a manual calculation of the ratios can be time‐consuming and labor‐intensive, especially when the number of peptides to be tested is large. To establish a liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry (LC/MALDI TOF/TOF)‐based targeted quantitative proteomics pipeline, we have developed a software named Mass Spectrometry based Quantification (MSQ). This software can be used to automate the quantification and identification of targeted peptides/proteins by the MALDI TOF/TOF platform. MSQ was applied to the detection of a selected group of targeted peptides in pooled human cerebrospinal spinal fluid (CSF) from patients with Alzheimer's disease (AD) in comparison with age‐matched control (OC). The results for the automated quantification and identification of targeted peptides/proteins in CSF were in good agreement with results calculated manually. Copyright © 2010 John Wiley & Sons, Ltd.     

Direct ampholyte-free liquid-phase isoelectric peptide focusing: application to the human serum proteome

In this study, we utilized a multidimensional peptide separation strategy combined with tandem mass spectrometry (MS/MS) for the identification of proteins in human serum. After enzymatically digesting serum with trypsin, the peptides were fractionated using liquid-phase isoelectric focusing (IEF) in a novel ampholyte-free format. Twenty IEF fractions were collected and analyzed by reversed-phase microcapillary liquid chromatography (microLC)-MS/MS. Bioinformatic analysis of the raw MS/MS spectra resulted in the identification of 844 unique peptides, corresponding to 437 proteins. This study demonstrates the efficacy of ampholyte-free peptide autofocusing, which alleviates peptide losses in ampholyte removal strategies. The results show that the separation strategy is effective for high-throughput characterization of proteins from complex proteomic mixtures.     

Comparison of data analysis parameters and MS/MS fragmentation techniques for quantitative proteome analysis using isobaric peptide termini labeling (IPTL)

Isobaric peptide termini labeling (IPTL) is a quantification method which permits relative quantification using quantification points distributed throughout the whole tandem mass spectrometry (MS/MS) spectrum. It is based on the complementary derivatization of peptide termini with different isotopes resulting in isobaric peptides. Here, we use our recently developed software package IsobariQ to investigate how processing and data analysis parameters can improve IPTL data. Deisotoping provided cleaner MS/MS spectra and improved protein identification and quantification. Denoising should be used with caution because it may remove highly regulated ion pairs. An outlier detection algorithm on the ratios within every individual MS/MS spectrum was beneficial in removing false-positive quantification points. MS/MS spectra using IPTL typically contain two peptide series with complementary labels resulting in lower Mascot ion scores than non-labeled equivalent peptides. To avoid this penalty, the two chemical modifications for IPTL were specified as variables including satellite neutral losses of tetradeuterium with positive loss for the heavy isotopes and negative loss for the light isotopes. Thus, the less dominant complementary ion series were not considered for the scoring, which improved the ion scores significantly. In addition, we showed that IPTL was suitable for fragmentation by electron transfer dissociation (ETD) and higher energy collisionally activated dissociation (HCD) besides the already reported collision-induced dissociation (CID). Notably, ETD and HCD data can be identified and quantified using IsobariQ. ETD outperformed CID and HCD only for charge states ≥4+ but yielded in total fewer protein identifications and quantifications. In contrast, the high-resolution information of HCD fragmented peptides provided most identification and quantification results using the same scan speed.     

Characterization of gingerol-related compounds in ginger rhizome (Zingiber officinale Rosc.) by high-performance liquid chromatography/electrospray ionization mass spectrometry

This study sought to determine the utility of liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) coupled with diode array detection in identifying gingerol-related compounds from crude extracts of ginger rhizome. The fragmentation behaviors of compounds in both (-)- and (+)ESI-MS/MS were used to infer and confirm the chemical structures of several groups of compounds, including the gingerols, methylgingerols, gingerol acetates, shogaols, paradols, gingerdiols, mono- and diacetyl gingerdiols, and dehydrogingerdiones. Diode array detection at different wavelengths was used to confirm MS/MS-based identification. In total, 31 gingerol-related compounds were identified from the methanolic crude extracts of fresh ginger rhizome in this study. Three of these compounds were found to be new compounds. This study demonstrated that LC/ESI-MS/MS is a powerful on-line tool for identification of gingerol-related compounds, especially for thermally labile compounds that cannot be readily detected by GC/MS analysis.     

Determination of ricin by nano liquid chromatography/mass spectrometry after extraction using lactose-immobilized monolithic silica spin column

Ricin is a glycosylated proteinous toxin that is registered as toxic substance by Chemical Weapons convention. Current detection methods can result in false negatives and/or positives, and their criteria are not based on the identification of the protein amino acid sequences. In this study, lactose-immobilized monolithic silica extraction followed by tryptic digestion and liquid chromatography/mass spectrometry (LC/MS) was developed as a method for rapid and accurate determination of ricin. Lactose, which was immobilized on monolithic silica, was used as a capture ligand for ricin extraction from the sample solution, and the silica was supported in a disk-packed spin column. Recovery of ricin was more than 40%. After extraction, the extract was digested with trypsin and analyzed by LC/MS. The accurate masses of molecular ions and MS/MS spectra of the separated peptide peaks were measured by Fourier transform-MS and linear iontrap-MS, respectively. Six peptides, which were derived from the ricin A-(m/z 537.8, 448.8 and 586.8) and B-chains (m/z 701.3, 647.8 and 616.8), were chosen as marker peptides for the identification of ricin. Among these marker peptides, two peptides were ricin-specific. This method was applied to the determination of ricin from crude samples. The monolithic silica extraction removed most contaminant peaks from the total ion chromatogram of the sample, and the six marker peptides were clearly detected by LC/MS. It takes about 5 h for detection and identification of more than 8 ng/ml of ricin through the whole handling, and this procedure will be able to deal with the terrorism using chemical weapon.     

A method for rapidly confirming protein N-terminal sequences by matrix-assisted laser desorption/ionization mass spectrometry

The N-terminal sequence is important for the identification of a protein and the confirmation of its N-terminal processing. Although mass spectrometry (MS) is a sensitive and high-throughput method to sequence and identify peptides and proteins, N-terminal peptides, diluted among most of the peptides that do not originate at the N-termini, are not easy to identify directly with MS. To develop a simple and rapid method to identify and sequence the N-terminal peptide of a protein, a new strategy based on specific sulfonation of terminal amino groups and selective monitoring of the sulfonated peptide was introduced. After a protein had been guanidinated, 2-sulfobenzoylated, and reduced, it was digested with trypsin and analyzed by MS. Because of the strong acidity of sulfonic groups and the specific sulfonation of alpha-amino groups, the sulfonated N-terminal peptide dominated as base peak in the negative mode peptide mass fingerprint (PMF) and was easy to identify. The N-terminal peptide was then selected as precursor ion for tandem mass spectrometric (MS/MS) analysis. Four proteins were tested with this method and their N-terminal peptides were successfully recognized and sequenced. The results suggest that the addition of a sulfonic acid group facilitates the identification and de novo sequencing of N-terminal peptides.     

Use of an integrated MS--multiplexed MS/MS data acquisition strategy for high-coverage peptide mapping studies

Liquid chromatography/mass spectrometry (LC/MS) peptide maps have become a basic tool for characterizing proteins of biological and pharmaceutical interest. The ability to generate reproducible maps with high protein sequence coverage is a central goal of methods development. We have applied a recently developed analytical approach (termed LC/MS(E)) to LC/MS peptide mapping. Using the LC/MS(E) approach, the mass detector alternates between a low-energy scanning mode (MS) for accurate mass peptide precursor identification, and an elevated-energy mode (MS(E)) for generation of accurate mass multiplex peptide fragmentation data. In this paper, we evaluate this analytical approach against a tryptic digest of yeast enolase. From the low-energy data, high peptide map coverage (98% of sequence from peptides >3 amino acids) was reproducibly obtained. The MS signal for essentially equimolar peptides varied over 2 orders of magnitude in intensity, and peptide intensities could be precisely and reproducibly measured. Using the temporal constraint that MS(E) peptide fragment ions exhibit chromatographic profiles that parallel the precursor ions that generated them, we were able to produce accurate mass time-resolved MS/MS information for all enolase peptides with sufficient abundance to produce a detectable fragment ion.     

Retention Characteristics of Peptides in RP-LC: Peptide Retention Prediction

A review of recent results of the use of chromatographic retention data in peptide identification and in the development of procedures for peptide retention prediction is presented. In recent years, reversed phase LC (RP-LC) has become an important tool in the separation of peptides in MS analysis. A challenging problem in a further expansion of RP-LC applications is the use of already available retention information for the identification purposes simultaneously with MS–MS identification. This overview focuses on the retention characteristics suggested in LC. We will discuss the application of the retention index concept in LC, which is widely used in GC to characterize retention of organic compounds. The use of retention indices as retention characteristics of analytes in LC was first suggested at the end of 1970s, however the application of retention indices is still somewhat rare today. There are several reasons for this. One is the relatively high sensitivity and variability of retention indices to the change of parameters of chromatographic systems. Another is the chemical restrictions in the search of the universal set of reference compounds suitable for retention scaling. Several methods were suggested for the prediction of the retention times of peptides. A frequently used approach is based on the additivity scheme and calculation of the elution time through the summation of retention coefficients of amino acids constituting the peptide. Such an approach allows fairly accurate predictions of the retention time of peptides made up of not more then 15–20 amino acid residues. Additional correction factors were suggested to improve predictions including corrections for the peptide length, peptide hydrophobicity, sequence of amino acids, etc. Suggested procedures are discussed in detail. Application of predicted retention times in the identification of peptides is considered. Current status of LC retention data collections is presented.     

Electrospray mass spectrometry in the clinical diagnosis of variant hemoglobins

A combination of mass spectrometric (MS) techniques [electrospray MS, liquid secondary ion MS (LSIMS) and MS-MS] has been used for variant hemoglobin (Hb) detection and characterization. Electrospray MS allowed analysis of mixtures of intact globins giving simultaneously the molecular weights (accuracy 1-2 Da) and information about relative amounts of globins present. Currently, 14 Da is the minimum molecular weight difference required experimentally to accurately measure different species present in a mixture of 15-16 kDa proteins. Thus 80 and 79% of the known variants of alpha and beta chains, respectively, can be detected in mixtures with their normal counterparts, including Hb S (molecular weight difference = 30 Da). Abnormal hemoglobins detected were fractionated by C4 reversed-phase high-performance liquid chromatography (HPLC), and the separated globin chains (or the mixture of whole precipitated globin) were digested by trypsin. The tryptic peptides were separated by C18 reversed-phase HPLC and analyzed by LSIMS to narrow down the mutation site to a single peptide. The mass measured in LSIMS frequently corresponded to a unique structure, thus giving the unequivocal identification of the mutation and its site. Where there was ambiguity, tandem MS on a Kratos Concept four-sector instrument was used for sequencing the abnormal peptide. The practical use of the methodologies presented is illustrated through analysis and identification of Hb G-San Jose, Hb Stanleyville II, Hb S and Hb Willamette.     

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.     

Fast analysis of low molecular mass compounds present in snake venom: identification of ten new pyroglutamate-containing peptides

Characterization of the peptide content in snake venoms can be an important tool for the investigation of new pharmacological lead compounds. For this purpose, single-step analysis of crude venoms has recently been demonstrated using mass spectrometry (MS) techniques. Reproducible profiles of ions in MS and MS/MS spectra may also be used to compare venoms from different species. In this work matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to obtain mass patterns of the major peptides (<8 kDa) found in pooled venoms from the genera Bothrops and Crotalus. Venoms from five different Bothrops species (B. jararaca, B. insularis, B. alternatus, B. jararacussu, and B. neuwiedi) and three Crotalus species (C. viridis, C. adamanteus and C. durissus terrificus) were analyzed. In agreement with other reports, venoms from Bothrops species contained a variety of peptides in the range m/z 1000-1500, and in some samples larger components (m/z 7000-8000) were detected. In the Crotalus species venoms were rich in peptides ranging from m/z 1000-1500 and 4000-5500. MS/MS experiments on the low molecular mass peptides (m/z 1000-1500) confirmed the presence of ten new bradykinin-potentiating peptides among venoms from genera Bothrops and Crotalus. In order to determine whether additional peptides could be identified after partial purification, B. jararaca venom was subjected to size-exclusion chromatography on Sephacryl S-200, and two distinct low molecular mass pools were analyzed further by MALDI-TOFMS. No additional peptides were detected from the pool with masses below 2000 Da but a substantial improvement with better resolution was observed for the pool with masses above 7000 Da, indicating that complex samples such as crude snake venoms can be analyzed for low molecular mass peptides using a single-step procedure.     

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.     

Site-specific N-glycosylation analysis: matrix-assisted laser desorption/ionization quadrupole-quadrupole time-of-flight tandem mass spectral signatures for recognition and identification of glycopeptides

The identification of glycosylation sites in proteins is often possible through a combination of proteolytic digestion, separation, mass spectrometry (MS) and tandem MS (MS/MS). Liquid chromatography (LC) in combination with MS/MS has been a reliable method for detecting glycopeptides in digestion mixtures, and for assigning glycosylation sites and glycopeptide sequences. Direct interfacing of LC with MS relies on electrospray ionization, which produces ions with two, three or four charges for most proteolytic peptides and glycopeptides. MS/MS spectra of such glycopeptide ions often lead to ambiguous interpretation if deconvolution to the singly charged level is not used. In contrast, the matrix-assisted laser desorption/ionization (MALDI) technique usually produces singly charged peptide and glycopeptide ions. These ions require an extended m/z range, as provided by the quadrupole-quadrupole time-of-flight (QqTOF) instrument used in these experiments, but the main advantages of studying singly charged ions are the simplicity and consistency of the MS/MS spectra. A first aim of the present study is to develop methods to recognize and use glycopeptide [M+H]+ ions as precursors for MS/MS, and thus for glycopeptide/glycoprotein identification as part of wider proteomics studies. Secondly, this article aims at demonstrating the usefulness of MALDI-MS/MS spectra of N-glycopeptides. Mixtures of diverse types of proteins, obtained commercially, were prepared and subjected to reduction, alkylation and tryptic digestion. Micro-column reversed-phase separation allowed deposition of several fractions on MALDI plates, followed by MS and MS/MS analysis of all peptides. Glycopeptide fractions were identified after MS by their specific m/z spacing patterns (162, 203, 291 u) between glycoforms, and then analyzed by MS/MS. In most cases, MS/MS spectra of [M+H]+ ions of glycopeptides featured peaks useful for determining sugar composition, peptide sequence, and thus probable glycosylation site. Peptide-related product ions could be used in database search procedures and allowed the identification of the glycoproteins.