Protein identification by in-gel digestion, high-performance liquid chromatography, and mass spectrometry: Peptide analysis by complementary ionization techniques

A biologically active protein fraction was isolated from rabbit intestine, purified by one-dimensional SDS-PAGE and stained with Coomassie Brilliant Blue. A predominant band of approximately 110-130 kDa was excised and digested in-gel with trypsin. The resulting peptides were extracted then separated by microbore reversed-phase high-performance liquid chromatography (HPLC). Mass spectrometric data from one HPLC fraction obtained by two different ionization techniques proved to be complementary. Matrix-assisted laser desorption/ionization (MALDI) showed nine peptide masses, which by post source decay analysis and database searching were attributed to two proteins. Nanoflow electrospray analysis performed on a hybrid tandem mass spectrometer of quadrupole-quadrupole-orthogonal acceleration time-of-flight (QqTOF) geometry detected six additional peptide components. On the basis of the additional peptides and superior quality collision-induced dissociation spectra typical of this instrument type, two further proteins were identified. The resolution afforded by the QqTOF instrument permitted charge state determination for the fragment ions while preserving the high detection sensitivity that was essential in obtaining the composition of this mixture of proteins.     

Sulfopeptide fragmentation in electron-capture and electron-transfer dissociation

Sulfopeptides can be misassigned as phosphopeptides because of the isobaric nature of the sulfo- and the phosphomoieties. Instruments having the ability to measure mass with high accuracy may be employed to distinguish these moieties based on their mass defect (the sulfo-group is 9 mmu lighter than the phosphomoiety). However, the assignment of the exact site(s) of post-translational modification is required to probe biological function. We have reported earlier that peptides with identical sequences containing either O-sulfo- or O-phospho-modifications display different fragmentation behavior (K. F. Medzihradszky et al., Mol. Cell. Proteom.2004, 3, 429-440). We have also established that O-sulfo moieties are susceptible to side-chain fragmentation during collision-induced dissociation. Our present study provides evidence that neutral SO(3) losses can also occur in electron capture dissociation and electron-transfer dissociation experiments. We also report that such neutral losses may be reduced by fragmenting peptide-alkali metal adducts, such as sodiated or potassiated peptides.     

Peptide sequence determination by matrix-assisted laser desorption ionization employing a tandem double focusing magnetic—Orthogonal acceleration time-of-flight mass spectrometer

This report describes the fragmentation processes for peptides induced by collisional activation of the ¹²C isobar of matrix-assisted laser desorption ionization (MALDI)-generated pseudomolecular ions employing an EBE orthogonal acceleration time-of-flight mass spectrometer and using xenon as the collision gas at a laboratory collision energy of 800 eV. These MALDI-collision-induced dissociation (CID) spectra are shown to provide sequence information of comparable quality to those obtained by using high energy CID conditions with liquid secondary ionization mass spectrometry on a four-sector tandem instrument. Peptide sequencing via MALDI-CID is demonstrated on three tryptic peptides obtained from a bacterial protein (P450 isozyme) of unknown sequence. Sensitivity is shown to be at the 1 pmol level for standard peptides.     

Fragmentation and sequencing of cyclic peptides by matrix-assisted laser desorption/ionization post-source decay mass spectrometry.

A series of synthetic cyclic decapeptides and other smaller cyclic peptides were analyzed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The investigated compounds were cyclized in a head-to-tail manner and contained non-proteinaceous amino acids, such as D-phenylalanine, D,L-4-carboxyphenylalanine, epsilon-aminocaproic acid, and gamma-aminobutyric acid, and were synthesized in a program to develop inhibitors of pp60(c-src) (Src), a tyrosine kinase that is involved in signal transduction and growth regulation. Post-source decay (PSD) spectra of the cyclic peptides featured abundant sequence ions. Two preferential ring opening reactions were detected resulting in linear fragment ions with an N-terminus of proline and a C-terminus of glutamic acid, respectively. MALDI-PSD spectra even permitted de novo sequencing of some cyclic peptides. Systematic studies on cyclic peptides using this method of fragmentation have not been reported to date. This work presents an easy mass spectrometric method, MALDI-PSD, for the characterization and identification of cyclic peptides.     

Glycan Side Reaction May Compromise ETD-Based Glycopeptide Identification

Tris(hydroxymethyl)aminomethane (Tris) is one of the most frequently used buffer ingredients. Among other things, it is recommended and is usually used for lectin-based affinity enrichment of glycopeptides. Here we report that sialic acid, a common ‘capping’ unit in both N- and O-linked glycans may react with this chemical, and this side reaction may compromise glycopeptide identification when ETD spectra are the only MS/MS data used in the database search. We show that the modification may alter N- as well as O-linked glycans, the Tris-derivative is still prone to fragmentation both in ‘beam-type’ CID (HCD) and ETD experiments, at the same time—since the acidic carboxyl group was ‘neutralized’—it will display a different retention time than its unmodified counterpart. We also suggest solutions that—when incorporated into existing search engines—may significantly improve the reliability of glycopeptide assignments.

Artifacts in four-sector tandem mass spectrometry

Several types of artifacts were shown to be present in 4-sector tandem collision-induced dissociation (CID) mass spectra. In CID spectra of protonated peptides produced by liquid secondary-ion mass spectrometry (LSIMS), peaks corresponding to successive losses of matrix molecules from the precursor ion were observed. In addition, peaks corresponding to MH+ ions of smaller peptides that were also present in the sample/matrix mixture in greater abundance than the selected precursor ion were observed. Both of these types of artifact peaks were shown to originate from the 'peak-at-every-mass' chemical noise at the same nominal mass as that selected by the first 2 sectors (MS1). These noise ions are transmitted through to the collision cell and produce fragments that are analysed and detected in the next 2 sectors (MS2). A second, unrelated, kind of artifact was found to be due to decompositions in the second field-free region of MS2 in an EBEB geometry machine. These artifacts, which are detectable over only a very limited mass range when using a conventional single-point detector, can be present over a much greater mass range when an array detector is used and when the collision cell is floated above ground potential. A clear understanding of the origins of all peaks in a CID spectrum is important in order to have a firm foundation for interpretation, manual or computer-aided, of the spectra of unknown compounds.     

Correlation between isoniazid resistance and superoxide reactivity in mycobacterium tuberculosis KatG

Isoniazid is an antituberculosis prodrug that requires activation by the catalase-peroxidase (KatG) of Mycobacterium tuberculosis. The activated species, presumed to be an isonicotinoyl radical, couples to NADH forming an isoniazid-NADH adduct that ultimately confers antitubercular activity. We have compared the catalytic properties of three KatGs associated with isoniazid resistance (resistance mutation KatGs, (RM)KatGs: R104L, H108Q, S315T) to wild-type enzyme and two additional lab mutations (wild-type phenotype KatGs, (WTP)KatGs: WT KatG, Y229F, R418L). Neither catalase nor peroxidase activities, nor the presence/absence of the Met-Tyr-Trp cross-link (as probed by LC/MS on tryptic digests of the protein), exhibited any correlation with isoniazid resistance. The yields of isoniazid-NADH adduct formed were determined to be 1-5, 4-12, and 20-70-fold greater for the (WTP)KatGs than the (RM)KatGs for the compound I, II, and III pathways, respectively, strongly suggesting a role for oxyferrous KatG (supported by superoxide consumption measurements) that correlates with drug resistance. Stopped-flow UV-visible spectroscopic studies revealed that all KatGs were capable of forming both compound II and III intermediates. Rates of compound II decay were accelerated 4-12-fold in the presence of isoniazid (vs absence) for the (WTP)KatGs but were unaffected by the drug for the (RM)KatGs. A mechanism for isoniazid resistance which accounts for the observed reactivity for each of the compound I, II, and III intermediates is proposed and suggests that the compound III pathway may be the primary factor in determining overall isoniazid resistance by specific KatG mutants, with secondary contributions arising from the compound I and II pathways.     

Characterization of protein iv-glycosylation by reversed-phase microbore liquid chromatography / electrospray mass spectrometry,complementary mobile phases,and sequential exoglycosidase digestion

A strategy for the identification of the site occupancy and glycoform heterogeneity, including sialylation occurring at specific sites of N-linked giycoproteins is presented using the asparagine-linked glycosylation on bovine fetuin for illustration. This is achieved by microbore high-performance liquid chromatography/electrospray ionization mass analysis (LC/ESIMS) of the tryptic glycopeptide mixtures with an acetonitrile-based mobile phase followed by sequential steps of residue (and linkage) specific glycoform degradation and LC/ESIMS analysis at each stage. In addition, chromatographic separation of the site-specific glycoforms of tryptic glycopeptides is accomplished by the use of an alternative, mass spectrometrically compatible mobile phase-water/ethanol/propanol/formic acid. By employing this nontraditional mobile phase for characterizing the complete tryptic digest, and using highly specific exoglycosidases in combination with LC/ESIMS analysis, a previously uncharacterized carbohydrate (a disialo biantennary complex oligosaccharide) was identified as a novel structure at Asn⁸¹ of bovine fetuin. (J Am Sot Mass Spectrom 1994, 5, 350-358)     

Partial De Novo Sequencing and Unusual CID Fragmentation of a 7 kDa,Disulfide-Bridged Toxin

A 7 kDa toxin isolated from the venom of the Texas coral snake (Micrurus tener tener) was subjected to collision-induced dissociation (CID) and electron-transfer dissociation (ETD) analyses both before and after reduction at low pH. Manual and automated approaches to de novo sequencing are compared in detail. Manual de novo sequencing utilizing the combination of high accuracy CID and ETD data and an acid-related cleavage yielded the N-terminal half of the sequence from the reduced species. The intact polypeptide, containing 3 disulfide bridges produced a series of unusual fragments in ion trap CID experiments: abundant internal amino acid losses were detected, and also one of the disulfide-linkage positions could be determined from fragments formed by the cleavage of two bonds. In addition, internal and c-type fragments were also observed.