Protein identification by in-gel digestion and mass spectrometric analysis

This protocol details a method for the identification of proteins that have been separated by gel electrophoresis. In-gel digestion of the protein bands with trypsin followed by quadrupole ion-trap or other triple quadrupole mass spectrometry techniques is described. The proteins can be identified by database searching of the mass fingerprint of the intact peptides and of the characteristic fragment masses produced by tandem mass spectrometry.     

A sequencing strategy for the localization of O-glycosylation sites of MUC1 tandem repeats by PSD-MALDI mass spectrometry

It is demonstrated with glycopeptides of the polymorphic epithelialmucin (MUC1) that post-source decay matrix-assisted laser desorptionionization (PSD-MALDI) is a fast, highly sensitive, and reproduciblemethod for the localization of O-glycosylation sites by reflectrontime-of-flight (TOF) mass spectrometry. We have analyzed GalNAc-carryingpeptides of up to 25 amino acids, and could distinguish evenneighboring glycosylation sites. This method was also able tolocalize and characterize disaccharides (e.g., the Thomsen-Friedenreichdisaccharide) on MUC1 derived peptides. PSD-MALDI-MS fragmention patterns were recorded in the positive ion mode from thesynthetic peptide TAP25 [(T^1aAPPAHGVT⁹S¹⁰APDT¹⁴RPAPGS²⁰) T^1bAPPA],an overlapping sequence of MUC1 tandem repeats, which was glycosylatedwith GalNAc in vitro. The glycosylation sites found were eitherThr9 or Thr1b in the monoglycosylated, Thr9 and Thr1b in thediglycosylated, and Thr9, Thr1b, and Ser20 in the triglycosylatedpeptide. A single PSD-MALDI-MS spectrum of the underivatizedand uncleaved di- or triglycosylated TAP25 peptide was sufficientto identify the glycosylation sites, thereby distinguishingsix potential, partly adjacent, glycosylation sites. The monoglycosylatedfraction was found to consist of a mixture of two glycosylatedspecies with the same molecular weight. This was shown by theanalysis of proteolytic digests. PSD-MALDI-MS of the resultingpeptides right out of the digestion probe was sufficient toidentify the GalNAc-glycosylation sites as either Thr9 or Thr1b,respectively. Beyond the methodical aspects the results revealedthat in vitro glycosylation of the TAP25 peptide with a transferasesystem from human milk differs from that obtained with a breastcancer cell transferase system. glycosylation sites O-glycosylation PSD-MALDI-MS MUC1 mucins     

Influenza A hemagglutinin C-terminal anchoring peptide: identification and mass spectrometric study

MALDI-TOF MS and N-terminal amino acid sequencing allowed us to identify several fragments of the C-terminal peptide of Influenza A hemagglutinin (HA) containing transmembrane domains (TMD). These fragments were detected in the organic phase of chloroform-methanol extracts from bromelain-treated virus particles. Heterogeneous fatty acylation of the C-terminus was revealed. Tritium bombardment technique might open an opportunity for 3D structural investigation of the HA TMD in situ.     

Tandem mass spectrometric method for definitive localization of phosphorylation sites using bromine signature

Determination of the phosphorylation site in peptides by conventional tandem mass spectrometry is subject to ambiguity due to the neutral loss of the phosphate groups, especially in multiphosphorylated peptides. To prevent the neutral loss, the phosphate groups in phosphoserine or phosphothreonine peptides were replaced by p-bromobenzyl mercaptan via β-elimination and Michael addition. The unique isotopic signature of the Br introduced facilitated definitive localization of phosphorylation sites in multiphosphorylated peptides with highly adjacent serine or threonine residues. This method could be used to confirm phosphorylation sites determined by conventional tandem mass spectrometry after phosphopeptide enrichment.     

Database analysis of O-glycosylation sites in proteins

Statistical analysis was carried out to study the sequential aspects of amino acids around the O-glycosylated Ser/Thr. 992 sequences containing O-glycosylated Ser/Thr were selected from the O-GLYCBASE database of O-glycosylated proteins. The frequency of occurrence of amino acid residues around the glycosylated Ser/Thr revealed that there is an increased number of proline residues around the O-glycosylation sites in comparison with the nonglycosylated serine and threonine residues. The deviation parameter calculated as a measure of preferential and nonpreferential occurrence of amino acid residues around the glycosylation site shows that Pro has the maximum preference around the O-glycosylation site. Pro at +3 and/or -1 positions strongly favors glycosylation irrespective of single and multiple glycosylation sites. In addition, serine and threonine are preferred around the multiple glycosylation sites due to the effect of clusters of closely spaced glycosylated Ser/Thr. The preference of amino acids around the sites of mucin-type glycosylation is found likely to be similar to that of the O-glycosylation sites when taken together, but the acidic amino acids are more preferred around Ser/Thr in mucin-type glycosylation when compared totally. Aromatic amino acids hinder O-glycosylation in contrast to N-glycosylation. Cysteine and amino acids with bulky side chains inhibit O-glycosylation. The preference of certain potential sequence motifs of glycosylation has been discussed.     

Surface glycoproteomic analysis of hepatocellular carcinoma cells by affinity enrichment and mass spectrometric identification

Cell surface glycoproteins are one of the most frequently observed phenomena correlated with malignant growth. Hepatocellular carcinoma (HCC) is one of the most malignant tumors in the world. The majority of hepatocellular carcinoma cell surface proteins are modified by glycosylation in the process of tumor invasion and metastasis. Therefore, characterization of cell surface glycoproteins can provide important information for diagnosis and treatment of liver cancer, and also represent a promising source of potential diagnostic biomarkers and therapeutic targets for hepatocellular carcinoma. However, cell surface glycoproteins of HCC have been seldom identified by proteomics approaches because of their hydrophobic nature, poor solubility, and low abundance. The recently developed cell surface-capturing (CSC) technique was an approach specifically targeted at membrane glycoproteins involving the affinity capture of membrane glycoproteins using glycan biotinylation labeling on intact cell surfaces. To characterize the cell surface glycoproteome and probe the mechanism of tumor invasion and metastasis of HCC, we have modified and evaluated the cell surface-capturing strategy, and applied it for surface glycoproteomic analysis of hepatocellular carcinoma cells. In total, 119 glycosylation sites on 116 unique glycopeptides were identified, corresponding to 79 different protein species. Of these, 65 (54.6?%) new predicted glycosylation sites were identified that had not previously been determined experimentally. Among the identified glycoproteins, 82?% were classified as membrane proteins by a database search, 68?% had transmembrane domains (TMDs), and 24?% were predicted to contain 2-13 TMDs. Moreover, a total of 26 CD antigens with 50 glycopeptides were detected in the membrane glycoproteins of hepatocellular carcinoma cells, comprising 43?% of the total glycopeptides identified. Many of these identified glycoproteins are associated with cancer such as CD44, CD147 and EGFR. This is a systematic characterization of cell surface glycoproteins of HCC. The membrane glycoproteins identified in this study provide very useful information for probing the mechanism of liver cancer invasion and metastasis.     

Sequence analysis of peptide mixtures by automated integration of Edman and mass spectrometric data.

A computer algorithm is described that utilizes both Edman and mass spectrometric data for simultaneous determination of the amino acid sequences of several peptides in a mixture. Gas phase sequencing of a peptide mixture results in a list of observed amino acids for each cycle of Edman degradation, which by itself may not be informative and typically requires reanalysis following additional chromatographic steps. Tandem mass spectrometry, on the other hand, has a proven ability to analyze sequences of peptides present in mixtures. However, mass spectrometric data may lack a complete set of sequence-defining fragment ions, so that more than one possible sequence may account for the observed fragment ions. A combination of the two types of data reduces the ambiguity inherent in each. The algorithm first utilizes the Edman data to determine all hypothetical sequences with a calculated mass equal to the observed mass of one of the peptides present in the mixture. These sequences are then assigned figures of merit according to how well each of them accounts for the fragment ions in the tandem mass spectrum of that peptide. The program was tested on tryptic and chymotryptic peptides from hen lysozyme, and the results are compared with those of another computer program that uses only mass spectral data for peptide sequencing. In order to assess the utility of this method the program is tested using simulated mixtures of varying complexity and tandem mass spectra of varying quality.     

Accurate peptide fragment mass analysis: multiplexed peptide identification and quantification

Fourier transform-all reaction monitoring (FT-ARM) is a novel approach for the identification and quantification of peptides that relies upon the selectivity of high mass accuracy data and the specificity of peptide fragmentation patterns. An FT-ARM experiment involves continuous, data-independent, high mass accuracy MS/MS acquisition spanning a defined m/z range. Custom software was developed to search peptides against the multiplexed fragmentation spectra by comparing theoretical or empirical fragment ions against every fragmentation spectrum across the entire acquisition. A dot product score is calculated against each spectrum to generate a score chromatogram used for both identification and quantification. Chromatographic elution profile characteristics are not used to cluster precursor peptide signals to their respective fragment ions. FT-ARM identifications are demonstrated to be complementary to conventional data-dependent shotgun analysis, especially in cases where the data-dependent method fails because of fragmenting multiple overlapping precursors. The sensitivity, robustness, and specificity of FT-ARM quantification are shown to be analogous to selected reaction monitoring-based peptide quantification with the added benefit of minimal assay development. Thus, FT-ARM is demonstrated to be a novel and complementary data acquisition, identification, and quantification method for the large scale analysis of peptides.     

Postexperiment monoisotopic mass filtering and refinement (PE-MMR) of tandem mass spectrometric data increases accuracy of peptide identification in LC/MS/MS

Methods for treating MS/MS data to achieve accurate peptide identification are currently the subject of much research activity. In this study we describe a new method for filtering MS/MS data and refining precursor masses that provides highly accurate analyses of massive sets of proteomics data. This method, coined "postexperiment monoisotopic mass filtering and refinement" (PE-MMR), consists of several data processing steps: 1) generation of lists of all monoisotopic masses observed in a whole LC/MS experiment, 2) clusterization of monoisotopic masses of a peptide into unique mass classes (UMCs) based on their masses and LC elution times, 3) matching the precursor masses of the MS/MS data to a representative mass of a UMC, and 4) filtration of the MS/MS data based on the presence of corresponding monoisotopic masses and refinement of the precursor ion masses by the UMC mass. PE-MMR increases the throughput of proteomics data analysis, by efficiently removing "garbage" MS/MS data prior to database searching, and improves the mass measurement accuracies (i.e. 0.05 +/- 1.49 ppm for yeast data (from 4.46 +/- 2.81 ppm) and 0.03 +/- 3.41 ppm for glycopeptide data (from 4.8 +/- 7.4 ppm)) for an increased number of identified peptides. In proteomics analyses of glycopeptide-enriched samples, PE-MMR processing greatly reduces the degree of false glycopeptide identification by correctly assigning the monoisotopic masses for the precursor ions prior to database searching. By applying this technique to analyses of proteome samples of varying complexities, we demonstrate herein that PE-MMR is an effective and accurate method for treating massive sets of proteomics data.     

Mass spectrometric identification of N- and O-glycosylation sites of full-length rat selenoprotein P and determination of selenide-sulfide and disulfide linkages in the shortest isoform

Rat selenoprotein P is an extracellular glycoprotein of 366 amino acid residues that is rich in cysteine and selenocysteine. Plasma contains four isoforms that differ principally by length at the C-terminal end. Mass spectrometry was used to identify sites of glycosylation on the full-length protein. Of the potential N-glycosylation sites, three located at residues 64, 155, and 169 were occupied, while the two at residues 351 and 356 were not occupied. Threonine 346 was variably O-glycosylated. Thus, full-length selenoprotein P is both N- and O-glycosylated. The shortest isoform of selenoprotein P, which terminates at residue 244, was analyzed for selenide-sulfide and disulfide linkages. In this isoform, a single selenocysteine and seven cysteines are present. Mass spectrometric analysis indicated that a selenide-sulfide bond exists between Sec40 and Cys43. Two disulfides were also detected as Cys149-Cys167 and Cys153-Cys156. The finding of a selenide-sulfide bond in the shortest isoform is compatible with a redox function of this pair that might be analogous to the selenol-thiol pair near the C terminus of animal thioredoxin reductase. The disulfide formed by Cys153-Cys156 also has some characteristics of a redox active pair.     

Direct mass spectrometric peptide profiling and fragmentation of larval peptide hormone release sites in Drosophila melanogaster reveals tagma-specific peptide expression and differential processing

Regulatory peptides represent a diverse group of messenger molecules. In insects, they are produced by endocrine cells as well as secretory neurones within the CNS. Many regulatory peptides are released as hormones into the haemolymph to regulate, for example, diuresis, heartbeat or ecdysis behaviour. Hormonal release of neuropeptides takes place at specialized organs, so-called neurohaemal organs. We have performed a mass spectrometric characterization of the peptide complement of the main neurohaemal organs and endocrine cells of the Drosophila melanogaster larva to gain insight into the hormonal communication possibilities of the fruit fly. Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and MALDI-TOF-TOF tandem mass spectrometry, we detected 23 different peptides of which five were unpredicted by previous genome screenings. We also found a hitherto unknown peptide product of the capa gene in the ring gland and transverse nerves, suggesting that it might be released as hormone. Our results show that the peptidome of the neurohaemal organs is tagma-specific and does not change during metamorphosis. We also provide evidence for the first case of differential prohormone processing in Drosophila.     

Gas chromatographic mass spectrometric identification of N-dicarboxylmonoglycines

A number of N-dicarboxylmonoglycines of biological interest have been synthesized. They were characterized by means of mass spectrometry. Gas chromatography of the methyl esters of methylmalonyl-, succinyl-, glutaryl-, adipyl-, suberyl- and sebacylglycines showed a single sharp peak for each compound on Dexsil 300 and OV-17 columns. Methylene unit values and mass spectra of the six methyl esters are reported.     

Mass spectrometric analysis of synapsins in Drosophila melanogaster and identification of novel phosphorylation sites

Synapsins are synaptic vesicle-associated phosphoproteins that play a major role in the fine regulation of neurotransmitter release. In Drosophila, synapsins are required for complex behavior including learning and memory. Synapsin isoforms were immunoprecipitated from homogenates of wild-type Drosophila heads using monoclonal antibody 3C11. Synapsin null mutants (Syn(97)) served as negative controls. The eluted proteins were separated by SDS-PAGE and visualized by silver staining. Gel pieces picked from five bands specific for wild type were analyzed by nano-LC-ESI-MS/MS following multienzyme digestion (trypsin, chymotrypsin, AspN, subtilisin, pepsin, and proteinase K). The protein was unambiguously identified with high sequence coverage (90.83%). A number of sequence conflicts were observed and the N-terminal amino acid was identified as methionine rather than leucine expected from the cDNA sequence. Several peptides from the larger isoform demonstrated that the in-frame UAG stop codon at position 582 which separates two large open reading frames is read through by tRNAs for lysine. Seven novel phosphorylation sites in Drosophila synapsin were identified at Thr-86, Ser-87, Ser-464, Thr-466, Ser-538, Ser-961, and Tyr-982 and verified by phosphatase treatment. No phosphorylation was observed at the conserved PKA/CaM kinase-I/IV site (RRFS, edited to RGFS) in domain A or a potential PKA site near domain E.     

Chemical cross-linking and mass spectrometric identification of sites of interaction for UreD, UreF, and urease

Synthesis of active Klebsiella aerogenes urease requires four accessory proteins to generate, in a GTP-dependent process, a dinuclear nickel active site with the metal ions bridged by a carbamylated lysine residue. The UreD and UreF accessory proteins form stable complexes with urease apoprotein, comprised of UreA, UreB, and UreC. The sites of protein-protein interactions were explored by using homobifunctional amino group-specific chemical cross-linkers with reactive residues being identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) of tryptic peptides. On the basis of studies of the UreABCD complex, UreD is capable of cross-linking with UreB Lys(9), UreB Lys(76), and UreC Lys(401). Furthermore UreD appears to be positioned over UreC Lys(515) according to decreased reactivity of this residue compared with its reactivity in UreD-free apoprotein. Several UreB-UreC and UreC-UreC cross-links also were observed within this complex; e.g. UreB Lys(76) with the UreC amino terminus, UreB Lys(9) with UreC Lys(20), and UreC Lys(515) with UreC Lys(89). These interactions are consistent with the proximate surface locations of these residues observed in the UreABC crystal structure. MALDI-TOF MS analyses of UreABCDF are consistent with a cross-link between the UreF amino terminus and UreB Lys(76). On the basis of an unexpected cross-link between UreB Lys(76) and UreC Lys(382) (distant from each other in the UreABC structure) along with increased side chain reactivities for UreC Lys(515) and Lys(522), UreF is proposed to induce a conformational change within urease that repositions UreB and potentially could increase the accessibility of nickel ions and CO(2) to residues that form the active site.     

Identification of enzymatically produced peptide fragments by liquid chromatography and mass spectrometry

The qualitative and quantitative determination of peptide fragments of angiotensin I generated by rat lung dipeptidyl carboxypeptidase (angiotensin converting enzyme, EC 3.4.15.1) is described. Enzymatically formed peptide fragments, after derivatization with fluorescamine, were separated and isolated by reverse-phase high-performance liquid chromatography. The recovered fluorescamine derivative of histidyl-leucine was then further identified by mass spectrometry. It is anticipated that this approach would be widely applicable to other enzyme systems.     

Effect of mass spectrometric parameters on peptide and protein identification rates for shotgun proteomic experiments on an LTQ-orbitrap mass analyzer

The success of a shotgun proteomic experiment relies heavily on the performance and optimization of both the LC and the MS systems. Despite this, little consideration has, so far, been given to the importance of evaluating and optimizing the MS instrument settings during data‐dependent acquisition mode. Moreover, during data‐dependent acquisition, the users have to decide and choose among various MS parameters and settings, making a successful analysis even more challenging. We have systematically investigated and evaluated the effect of enabling and disabling the preview mode for FTMS scan, the number of microscans per MS/MS scan, the number of MS/MS events, the maximum ion injection time for MS/MS, and the automatic gain control target value for MS and MS/MS events on protein and peptide identification rates on an LTQ‐Orbitrap using the Saccharomyces cerevisiae proteome. Our investigations aimed to assess the significance of each MS parameter to improve proteome analysis and coverage. We observed that higher identification rates were obtained at lower ion injection times i.e. 50–150 ms, by performing one microscan and 12–15 MS/MS events. In terms of ion population, optimal automatic gain control target values were at 5×10⁵–1×10⁶ ions for MS and 3×10³–1×10⁴ ions for MS/MS. The preview mode scan had a minimal effect on identification rates. Using optimized MS settings, we identified 1038 (±2.3%) protein groups with a minimum of two peptide identifications and an estimated false discovery rate of ～1% at both peptide and protein level in a 160‐min LC‐MS/MS analysis.