Javier Gonzalez

A method for determination of N-glycosylation sites in glycoproteins by collision-induced dissociation analysis in fast atom bombardment mass spectrometry: Identification of the positions of carbohydrate-linked asparagine in recombinant α-amylase by treatment with peptide-N-glycosidase F in 18O-labeled water

Previously, a combined use of fast atom bombardment (FAB) mass spectrometry and peptide N-glycosidase F, an enzyme that cleaves the beta-aspartylglycosylamine linkage of Asn-linked carbohydrates, was successfully applied to identification of N-glycosylation sites in a glycoprotein with the known or DNA-derived sequence (S. A. Carr and G. D. Roberts, 1986, Anal. Biochem. 157, 396-406). Here, we extended the method for easier identification of N-glycosylation sites in a glycoprotein even with unknown sequence. The glycoprotein is digested with peptide-N-glycosidase F in buffer containing 40 at% H2 18O, to yield a deglycosylated protein whose carbohydrate-linked Asn residues are converted to Asp partly labeled with 18O at their beta-carboxyl group during this digestion. The deglycosylated protein is further digested with proteolytic enzymes in an appropriate buffer prepared with normal water, and then peptides are separated on a reversed-phase column by HPLC. Peptides in which carbohydrate-linked Asn has been converted to Asp show a pair of signals ([M + 1]+ and [M + 3]+) in FAB mass spectra due to the partial incorporation of 18O into the beta-carboxyl groups of Asp residues, while the other peptides show normal isotopic ion distributions. Thus, both formally N-glycosylated peptides and, using collision-induced dissociation analysis, N-glycosylation sites can be identified. The application of the present method to the determination of N-glycosylation sites in a recombinant glycoprotein, Bacillus licheniformis alpha-amylase, is described.

Automated interpretation of low-energy collision-induced dissociation spectra by SeqMS, a software aid for de novo sequencing by tandem mass spectrometry

SeqMS, a software aid for de novo sequencing by tandem mass spectrometry (MS/MS), which was initially developed for the automated interpretation of high‐energy collision‐induced dissociation (CID) MS/MS spectra of peptides, has been applied to the interpretation of low‐energy CID and post‐source decay (PSD) spectra of peptides. Based on peptide backbone fragmented ions and their related ions, which are the dominant ions observed in the latter two techniques, the types of ions and their propensities to be observed have been optimized for efficient interpretation of the spectra. In a typical example, the modified SeqMS allowed the complete sequencing of a 31‐amino acid synthetic peptide, except for the isobaric amino acids (Leu or Ile, and Lys or Gln), based on only the low‐energy CID‐MS/MS spectrum.

Effect of the position of a basic amino acid on C-terminal rearrangement of protonated peptides upon collision-induced dissociation

Internal rearrangement involving the loss of the C-terminal amino acid residue upon collision-induced dissociation (CID) or metastable decomposition was studied for protonated peptides. To investigate the structural characteristics of peptides responsible for this rearrangement, a series of synthetic peptides were prepared and subjected to B/E-linked scan or tandem mass spectrometric analyses using a four-sector instrument. The results showed that the position of a basic amino acid in the peptide sequence and its basicity have a significant influence on the rearrangement. Arginine (Arg) located at the n-1 position facilitates the rearrangement with about twice as many rearrangement ions as is observed for the other Arg-containing peptides. This can be attributed to the interaction of a positively charged guanidino group of Arg with its own carbonyl group via a salt bridge which is tightly formed in vacuo between a guanidino and carboxylate groups, the mechanism of which is analogous to that previously proposed for the formation of similar rearrangement ions observed in the spectra of metal-cationized peptides. This association would result in the facile attack of the C-terminal hydroxyl group on the penultimate carbonyl group, leading to the rearrangement. In addition, the rearrangement ion was observed both in metastable decomposition and high-energy CID spectra obtained by B/E-linked scan analyses without or with gas, respectively, but in a sequence dependent manner.

Automated interpretation of high-energy collision-induced dissociation spectra of singly protonated peptides by ‘seqms', a software aid for de novo sequencing by tandem mass spectrometry

SeqMS, a software program designed for the automated interpretation of high-energy collision-induced dissociation (CID) mass spectra of singly protonated peptides ionized by fast atom bombardment, has been developed. The software is capable of probing the sequence of an unknown peptide, and even of certain modified peptides. The program, compiled for WINDOWS95 or NT, also permits the retrieval of raw data and the reconstruction of the spectra on a user-friendly graphical interface with the aid of several tools for processing the spectra, which include setting multiple threshold levels and automatic peak detection. SeqMS is capable of generating candidate sequences, based on the detected peaks, and of displaying the resulting assignments for each candidate in a spectrum or in tabular form. The software has the following capabilities: 1) the ions derived from backbone and side-chain fragmentations, internal and immonium ions, and side-chain loss ions can be used for calculation; 2) 18O-labeling of a peptide at the C terminus, a methodology which was developed to differentiate N-terminal from C-terminal ions, is applicable as an optional setting; 3) modified amino acids and N- or C-terminal blocking groups are taken into account for calculation according to the users setting in a library; 4) amino acid composition and partial or complete amino acid sequence of a peptide can be used as input for calculation; 5) the assignments of signal output in a spectrum can be graphically edited, and then re-calculated based on the edited peaks. The efficacy of the program is demonstrated by testing 74 high-energy CID spectra, obtained using a four-sector instrument, of synthetic, proteolytic, and biologically active peptides, some of which contain modified groups.

A new type of mitogenic factor produced by Streptococcus pyogenes

A new type or mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP‐Sephadex C‐25 column chromatography, preparative isoelectric focusing and reversed‐phase high‐performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single‐band staining for protein an SDS‐PAGE, The molecular weight of the purified mitogenic factor was determined to be 25.370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N‐terminal region of the purified mitogenic factor was determined to be Gin‐Thr‐Gin‐Val‐Ser‐Asn‐Asp‐Val‐Val‐Leu‐Asn‐Asp‐Gly‐Ala‐Ser‐Lys‐Tyr‐Leu‐Asn‐Glu‐Ala‐, which was also different from the reported N‐terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.

A computer program to aid the sequencing of peptides in collision-activated decomposition experiments

A computer program named MSEQ, based on graph theory has been implemented to aid the sequencing of peptides from collision-activated decomposition (CAD) spectra. Input data required by this program are: the molecular weight of the peptide, the list of the masses of the daughter ions and the masses of the N- and C-terminal groups. The output comprises a list of the most likely sequences with their respective scores and the assignments of the daughter ions. A set of probabilities for each fragment ion was computed from hundreds of CAD spectra obtained from our mass spectrometer. To date many peptides have been sequenced in our laboratory with the help of this program, and in most of them the real sequence ranks among the five top sequences. The program is able to differentiate isobaric amino acids such as leucine and isoleucine when the side-chain fragmentation appears in the spectrum. A criterion is used to discard those sequences that match the spectrum poorly from the earliest steps. The program is fast and consumes no memory.