Sequencing of anti-thyroxine monoclonal antibody fab fragment by ion trap mass spectrometry

A comprehensive mass spectrometric strategy is described for the sequencing of anti-thyroxine monoclonal antibody Fab region (48 000 Da). After reduction and S-carboxymethylation of the Fab, the modified light chain and Fd fragment were separated and subjected to multiple proteolytic digestions. The resulting digests were characterized by on-line microbore liquid chromatography/electrospray ionization ion trap mass spectrometry. Database search against published immunoglobulins (IgGs) allowed identification of all the peptides in constant domains. The homologous framework residues in the IgGs were utilized as 'sequence maps' for the sequence determination of variable domains. S-Carboxymethylation with an isotopic-enriched moiety greatly facilitated the recognition and data elucidation of cysteinyl peptides through the unique isotopic distribution patterns specific to the modified peptides. Methylation of peptide mixtures provided additional information for the interpretation of MS/MS spectra, allowing easy differentiation of Asp/Asn and Gln/Glu pairs. This study clearly demonstrates the power of mass spectrometry for the sequencing of antibodies without knowing the corresponding DNA sequences.     

Characterization of bovine surfactant proteins B and C by electrospray ionization mass spectrometry

Bovine surfactant proteins B (SP-B) and C (SP-C) were analyzed by nano-electrospray ionization mass spectrometry (nano-ESI-MS). The observed molecular masses showed discrepancies compared to the calculated molecular masses using the published amino acid sequences. The number of cysteine residues in the published bovine SP-B amino acid sequences also failed to match the observed mass shift upon reduction of the SP-B dimer. To determine the amino acid sequences of two proteins, SP-B was first digested with trypsin and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS), while SP-C was analyzed by MS/MS in its intact form. The amino acid sequence of bovine SP-B determined here matches the observed molecular mass. The sequence is almost identical to the sheep SP-B except for two amino acid residues, consistent with the proximity of the two species. The correct sequence contains seven cysteine residues. Bovine SP-B exists as dimers and all cysteines are oxidized to form disulfide bonds in physiological conditions, which is in agreement with the observed mass shift upon reduction of the SP-B dimer. These cysteine residues are completely conserved across all species indicating their importance for the biological functions of this surfactant protein. The sequence of SP-C determined here also reveals an L to V substitution at its position 22 compared with the published bovine SP-B sequence.     

Characterization of a cross-linked DNA-Endonuclease VIII repair complex by electrospray ionization mass spectrometry

Electrospray mass spectrometry techniques were used to characterize components of the active site in Endonuclease VIII by identifying the amino acid sequence and the binding site for a tryptic peptide derived from Endo VIII in a cross-linked DNA-peptide complex. Endo VIII, a DNA repair enzyme with both glycosylase and lyase activities, was covalently bound to a thymidine glycol-containing oligodeoxynucleotide duplex by converting a transient Schiff base formed during the course of the glycosylase activity to a stable covalent bond by chemical reduction with sodium borohydride. After tryptic digestion of the initial product, the identification of the cross-linked peptide was deduced initially from the molecular mass of the tryptic product obtained by negative ion electrospray mass analysis. Nanospray tandem mass spectrometry (MS/MS) analysis of the tryptic product corroborated the molecular mass of the peptide fragment and verified the point of attachment to the oligomer, but failed to produce sufficient fragmentation to sequence the peptide completely. Direct evidence for the amino acid sequence of the peptide was obtained after enzymatic digestion of the DNA portion of the cross-linked DNA-peptide product and analysis by negative ion nanospray MS/MS. Examination of the ions from collision induced fragmentation disclosed that this substance was the N-terminal tryptic fragment of Endo VIII cross-linked to a portion of the oligomer, and that the N-terminal proline from Endo VIII was covalently bound to the residual deoxyribose moiety at the original location of the thymine glycol in the oligomer.     

High-resolution electrospray ionization fourier transform mass spectrometry with infrared multiphoton dissociation of glucokinase from Bacillus stearothermophilus

Glucokinase (GK, EC 2.7.1.2), a member of the enzyme family of hexokinases, has been shown to be linked to maturity-onset diabetes of the young type II (MODY-2). Although nucleotide and amino acid sequence information are available for the human varieties, they are not known for the variety from Bacillus stearothermophilus, which is often used in protein binding studies. Here, a combination of electrospray Fourier transform mass spectrometry (FTMS) and infrared multiphoton dissociation (IRMPD) is used to obtain accurate molecular weight and preliminary amino acid sequence information for the protein. Electrospray FTMS provides evidence of a solution phase dimer. In addition, dithiothreitol reduction shows no shift in high-resolution isotopic distributions, indicating a probable absence of disulfide bonds in the protein. The partial sequence information obtained from IRMPD could be the basis for creating a DNA probe for the protein.     

Determination of N-linked glycosylation of yeast external invertase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The extent of N-glycosylation of yeast external invertase at each of the 14 potential sites was determined by the combination of proteolytic digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). The average molecular mass of the intact external invertase was determined as 97 kDa by MALDI/TOF-MS. The intact protein was digested with trypsin, Lys-C and Asp-N, followed by high-performance liquid chromatographic separation. The proteolytic digests were analyzed by MALDI/MS screening for the glycopeptides. The glycopeptides were then treated with peptide:N-glycosidase F (PNGase F) and/or endo-beta-N-acetylglucosaminidase (Endo H) and the molecular mass of the deglycosylated peptide was determined by MALDI/MS and matched with the peptide predicted by a computer program. The sequences of some peptides or deglycosylated peptides were identified by the MALDI post-source decay technique. The size of the oligosaccharide, the degree of glycosylation and the distribution of the oligosaccharides at each individual potential glycosylation site were characterized. This information goes for beyond previously published data and sometimes differs from them. During this study, the amino acid sequence originally derived from the DNA sequence of the gene coding for invertase was also verified and it was found that this protein when expressed from SUC2 gene might be created as more than one sequence which differ by a few amino acid substitutions (Asn58<-->Thr, Asn65-->His and Val412<-->Ala).     

Identification of amino acid thiohydantoin derivatives by chemical ionization mass spectrometry

The chemical ionization mass spectra of 16 amino acid thiohydantoins were examined using isobutane or ammonia as reagent gases. Except for a few cases, including some aromatic amino acids, the chemical ionization spectra were much simpler than the corresponding electron impact spectra. Therefore, the major component in the amino acid thiohydantoin mixture was easily detected by chemical ionization mass spectrometry. The combination of the chemical ionization method and thiohydantoin formation was applied successfully to the sequence analysis of model peptides.     

Electrospray ionization tandem mass spectrometry sequencing of novel skin peptides from Ranid frogs containing disulfide bridges

Tandem mass spectrometry sequencing, as well as Edman sequencing of peptides belonging to the Rana genus, represents a difficult task due to the presence of a disulfide bridge at the C-terminus and their rather high molecular masses (over 2000 Da). The present study throws light upon the sequence of three rather long peptides (more than 20 amino acid residues each) isolated from the skin secretion of Russian frogs, Rana ridibunda and Rana arvalis. This novel aspect involves the fact that the sequences (including two sequences established de novo) were determined exclusively by means of mass spectrometry. A combination of electron capture dissociation (ECD) and collision-induced dissociaiton (CID) data accompanied by exact mass measurements (LTQ Fourier transform ion cyclotron resonance mass spectrometer) facilitated reaching the goal. To overcome the difficulty dealing with disulphide bridges ("Rana box"), reduction of the S-S bond with dithiotreitol followed by derivatization of Cys residues with iodoacetamide was used. The sequence was determined using combined spectral data on y and b series of fragment ions. A multiple mass spectrometry (MS(3)) experiment was also used to elucidate the sequence inside the "Rana box" after cysteine derivatization. Exact mass measurements were used to differentiate between Lys and Gln residues, while characteristic losses of 29 and 43 Da (d and w fragment ions) in CID and ECD experiments allowed us to distinguish between Ile and Leu isomeric acids.     

Identification of isoenzymes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The identification of isoforms is one of the great challenges in proteomics due to the large number of identical amino acids preventing their separations by two-dimensional electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has become a rapid and sensitive tool in proteomics, notably with the new instrumental improvements. In this study, we used several acquisition modes of MALDI-TOFMS to identify isoforms of porcine glutathiones S-transferase. The use of multiple proteases coupled to the different acquisition modes of MALDI-TOFMS (linear, reflectron, post-source decay (PSD) and in-source decay, positive and negative modes) allowed the identification of two sequences. Moreover, a third sequence is pointed out from a PSD study of a tryptic ion revealing the modification of the amino acid tyrosine 146 to phenylalanine.     

Amino-catalyzed fragmentation characteristics of polyamides containing N-methylpyrrole and N-methylimidazole by electrospray negative ionization mass spectrometry

Different negative fragmentations of synthesized polyamides containing N-methylpyrrole and N-methylimidazole were analyzed by electrospray ionization tandem mass spectrometry with low-energy collision-induced dissociation. An interesting rearrangement derived from amino catalysis of N-methylimidazole was observed. The observation is useful for the study of the correlations between the sequences of polyamides and their mass fragmentation pathways.     

Structural studies of the allelic wheat glutenin subunits 1Bx7 and 1Bx20 by matrix-assisted laser desorption/ionization mass spectrometry and high-performance liquid chromatography/electrospray ionization mass spectrometry

Structural studies of the high molecular mass (HMM) glutenin subunits 1Bx7 (from cvs Hereward and Galatea) and 1Bx20 (from cv. Bidi17) of bread wheat were conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). For all three proteins, MALDI-TOFMS analysis showed that the isolated fractions contained a second component with a mass about 650 Da lower than the major component. The testing and correction of the gene-derived amino acid sequences of the three proteins were performed by direct MALDI-TOFMS analysis of their tryptic peptide mixture. Analysis of the digest was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, optimizing the matrix and the acquisition parameters for each mass range. Complementary data were obtained by RP-HPLC/ESI-MS analysis of the tryptic digest. This resulted in coverage of about 98% of the sequences. In contrast to the gene-derived data, the results obtained demonstrate the insertion of the sequence QPGQGQ between Trp716 and Gln717 of subunit 1Bx7 (cv. Galatea) and a possible single amino acid substitution within the T20 peptide of subunit 1Bx20. Moreover, the mass spectrometric data demonstrated that the lower mass components present in all the fractions correspond to the major components but lack about six amino acid residues, which are probably lost from the protein C-terminus. Finally, the results obtained provide evidence for the lack of glycosylation or other post-translational modifications of these subunits.     

纳升级电喷雾离子源-四极杆-飞行时间质谱中磷酸化肽的离子抑制作用

目前磷酸化蛋白质组学研究中的主要技术是蛋白质酶解产生的磷酸化肽的质谱检测。但是实际样品中的磷酸化肽(特别是多磷酸化肽)很难被检测到。其原因普遍认为是由于质谱检测时,非磷酸化肽抑制磷酸化肽。但也有认为非磷酸化肽对磷酸化肽没有抑制作用。另外磷酸化肽之间是否存在离子抑制作用还没有报道。本文采用相同氨基酸序列的标准磷酸化肽和非磷酸化肽,将其单独和混合进行质谱检测,通过对比混合前后磷酸化肽的信号强度,证明了非磷酸化肽对磷酸化肽有离子抑制作用;单磷酸化肽对二磷酸化肽有一定的抑制作用,但不太显著;单磷酸化肽对三磷酸化肽、二磷酸化肽对三磷酸化肽均有显著的离子抑制作用。该研究为今后单磷酸化肽和多磷酸化肽的分段富集和检测提供了有力的证明。     

Novel phosphoryl derivatization method for peptide sequencing by electrospray ionization mass spectrometry

A one-step phosphoryl derivatization method has been used in a peptide sequencing procedure for electrospray ionization tandem mass spectrometry (ESI-MS/MS). The sodiated derivatized peptides exhibit very simple dissociation patterns, in which two kinds of fragment ions, [b(n) + OH + Na]+ and [a(n) + Na]+, are formed. Since the amino acid residues are lost sequentially from the C-terminus, peptide sequences can be identified easily. The fragmentation efficiency of peptides increased as a result of the phosphorylation, and also provided peaks of useful intensity at lower m/z. A peptide with lysine at the C-terminus was derivatized and analyzed by ESI-MS/MS. Similar mass spectra, from which the sequence could be read out, were obtained. This is a novel derivatization method yielding neutral derivatives that should be suitable for peptide sequencing by LC/ESI-MS/MS.     

Characterization of amino acid‐derived betaines by electrospray ionization tandem mass spectrometry

Betaines belong to the naturally occurring osmoprotectants or compatible solutes present in a variety of plants, animals and microorganisms. In recent years, metabolomic techniques have been emerging as a fundamental tool for biologists because the constellation of these molecules and their relative proportions provide with information about the actual biochemical condition of a biological system. Therefore, identification and characterization of biologically important betaines are crucial, especially for metabolomic studies. Most of the natural betaines are derived from amino acids and related homologues. Although, theoretically, all the amino acids can be converted to corresponding betaines by simple methylation of the amine group, only a few of the amino acid‐derived betaines were fully characterized in the literature. Here, we report a combined electrospray ionization tandem and high‐resolution mass spectrometry study of all the betaines derived from amino acids, including the isomeric betaines. The decomposition pathway of protonated, sodiated and potassiated molecule ions that enable unambiguous characterization of the betaines including the isomeric betaines and overlapping ionic species of different betaines is distinctive. Copyright © 2012 John Wiley & Sons, Ltd.     

Unambiguous characterization and tissue localization of Pru P 3 peach allergen by electrospray mass spectrometry and MALDI imaging

The lipid transfer protein (LTP), Pru p 3, has been identified as the major allergen present in peach, and its sequence obtained by direct amino acid sequencing has been previously reported. However, several sequences, obtained from c‐DNA and available in databases, show differences among them and from the originally proposed structure. In this paper, we report the fast and unambiguous determination of the structure of Pru p 3 protein, extracted from three different varieties of peach, by electrospray ionization mass spectrometry (ESI‐MS), both coupled to single stage (quadrupole) or advanced (FT‐HRMS) analyzers. The structure was identical to one of the cDNA‐derived sequences and different in two positions from the previously reported structure obtained by amino acid sequencing. Moreover, the exclusive localization of the protein in the outer part of the fruits was assessed by Matrix‐Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI MSI). The results reported here demonstrate the full potential of mass spectrometry for rapidly obtaining high quality structural data of relevant food proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Zinc-finger motif noncovalent interactions with double-stranded DNA characterized by negative-ion electrospray ionization mass spectrometry

A zinc-finger motif recognizes specific sequences on the double helical structure of DNA. This sequence recognition property offers great promise for various biotechnology applications. Accordingly, it is crucially important to characterize zinc-finger binding characteristics for further developments. Although the gel shift assay or phage display is traditionally used for determining the binding characteristics of zinc-fingers for double stranded DNA, in the present study we utilize electrospray ionization mass spectrometry as an advanced and convenient characterization tool because of the rich information it provides, and its quantitative sensitivity, operational simplicity, and no need for radioactive labeling. Here we demonstrate the use of negative-ion electrospray ionization mass spectrometry for competition-based quantitative comparison of the zinc-finger motif sequence specificity, stoichiometry, and metal ion dependence.     

Fast protein sequence determination with matrix-assisted laser desorption and ionization mass spectrometry

Measurement and identification of digested peptides by matrix-assisted laser desorption and ionization mass spectrometry (LDI-MS) is demonstrated. Synthetic human parathyroid hormone, pTH (1-34), with a molecular mass of 4117.8 Da was digested with carboxypeptidases Y and B and the sequence of 14 amino acids from the C-terminus of the peptide was determined by analyzing the molecular mass of the truncated peptides. Furthermore, a tryptic digestion of pTH (1-34) was carried out and a molecular mass map of pTH (1-34) was obtained. With the results of the proteolytic digestion a rapid confirmation of the amino-acid sequence of the protein was possible. It is shown that the results of the tryptic digestion can be used for the unambiguous identification of the amino acid residues Lys and Arg, which cannot be distinguished with a mass spectrometer because of their equal nominal masses. Several advantages of amino acid sequence determination by the combination of digestion and LDI-MS are obvious: high sensitivity in the low pmol range, fast digestion time due to high enzyme/substrate ratios, quantification is unnecessary because the amino acids are identified by their molecular mass differences, the low chemical expenditure for the digestions and the accuracy of the sequence determination. Measurements with LDI-MS are fast: sample preparation and the measurement take only a few min. The mass determination and amino acid sequence is completely unimpaired by amino acid contaminations or impurities in the sample. The sensitivity of the method is in the low pmol to fmol range and thus comparable to other analytical methods.     

Screening of amino acids in dried blood spots by stable isotope derivatization-liquid chromatography-electrospray ionization mass spectrometry

Direct infusion mass spectrometry (DIMS) is a powerful technique in clinical diagnosis for screening neonatal amino acid metabolic disorders from dried blood spots (DBS). However, DIMS sometimes generated false-positive results for analysis of amino acids. In this work, we utilized a stable isotope derivatization method, combining with liquid chromatography tandem mass spectrometry (SID-LC-MS), to improve the specificity for screening amino acids in DBS specimens. A pair of isotope reagents, p-(dimethylamino)phenyl isothiocyanate (DMAP-NCS) and 4-isothiocyanato-N,N-bis(methyl-[²H₂])aniline ([²H₄]DMAP-NCS), was synthesized and used to label amino acids in DBS specimens. The [²H₄]DMAP-NCS labelled amino acid standards were used as internal standards to compensate the matrix effect. This method was validated by measuring linearity, recovery and accuracy. The results showed that the developed SID-LC-MS method can be used for sensitive and selective determination of 12 diagnostically important amino acids in DBS specimens.     

Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry

A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide and for phosphoserine-containing peptides derived from beta-casein and ovalbumin was the beta-elimination of phosphoric acid with concomitant conversion of phosphoserine to dehydroalanine and phosphothreonine to 2-aminodehydrobutyric acid. Peptide fragment ions of the b- and y-type allowed, in all cases, the localization of phosphorylation sites. Ion signals corresponding to (b-17), (b-18) and (y-17) fragment ions were also observed. The abundant neutral loss of phosphoric acid (-98 Da) is useful for femtomole level detection of phosphoserine-peptides in crude peptide mixtures generated by gel in situ digestion of phosphoproteins.     

Water cluster calibration reduces mass error in electrospray ionization mass spectrometry of proteins

Herein we report a novel calibration routine for use in positive ion mode electrospray ionization mass spectrometry (ESI-MS). Monoisotopic masses were calculated for a series of water clusters and used as calibration reference files (available at http://www.hbcg.utmb.edu/xray). The water cluster series contains singly charged peaks every 18 Da, which allows calibration curves to be precisely defined over a broad mass-to-charge ratio range. Water clusters, induced by a combination of high flow rate and high cone voltage, were used to accurately calibrate a quadrupole mass spectrometer from 100 to 1900 m/z. Calibration curves thus generated have many more data points and greatly reduced standard deviations compared to those obtained from myoglobin, sodium iodide, cesium iodide, or poly(ethylene glycol) based calibration standards. This calibration routine reduces the error in protein mass measurements by a factor of 3, from ±0.01% to ±0.0035% at the 95% confidence limit. The implications of this increased mass accuracy and wider calibrated mass-to-charge ratio scale for the study of protein sequence, structure, and folding by ESI-MS are discussed.     

Characterisation of botulinum toxins type A and B,by matrix-assisted laser desorption ionisation and electrospray mass spectrometry

A method earlier developed for the mass spectrometric (MS) identification of tetanus toxin (TTx) was applied to botulinum toxins type A and B (BTxA and BTxB). Botulinum toxins are extremely neurotoxic bacterial toxins, likely to be used as biological warfare agent. Biologically active BTxA and BTxB are comprised of a protein complex of the respective neurotoxins with specific haemagglutinins (HAs) and non-toxic non-haemagglutinins (NTNHs). These protein complexes are also observed in mass spectrometric identification. The particular BTxA complex, from Clostridium botulinum strain 62A, almost completely matched database data derived from genetic sequences known for this strain. Although no such database information was available for BTxB, from C. botulinum strain okra, all protein sequences from the complex except that of HA-70 were found to match proteins known from other type B strains. It was found that matrix-assisted laser desorption ionisation MS provides provisional identification from trypsin digest peptide maps and that liquid chromatography electrospray (tandem) mass spectrometry affords unequivocal identification from amino acid sequence information of digest peptides obtained in trypsin or pepsin digestion.