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Galactosamine was found consistently as a minor component of the envelope of five species of slow-growing mycobacteria, including all the major human pathogens, but not three rapid-growing species. The amino sugar was a component of the arabinogalactan of the cell wall skeleton, and occurred at the level of about one residue per arabinogalactan chain. Its amino group was in the free, un-N-acetylated state. Examination of oligosaccharides released by partial acid hydrolysis of arabinogalactan by fast atom bombardment-MS and gas chromatography-MS identified a series of oligoarabinans, each possessing one GalN unit, linked to position 2 of arabinose. It is proposed that the GalN residues occur as stub branches of 1-->5-linked arabinose chains in the arabinogalactan. Possible functions of GalN are discussed.

Pili, which are filamentous protein structures on the surface of the meningitis-causing organism Neisseria meningitidis, are known to be post-translationally modified with substituents that affect their mobility in SDS/PAGE and which might play a crucial role in adherence and bloodstream invasion. Tryptic digests of pili were analysed by fast atom bombardment and electrospray MS to identify putative modifications. Serine-93 was found to carry a novel modification of alpha-glycerophosphate. This is the first time that alpha-glycerophosphate has been observed as a substituent of a prokaryotic or eukaryotic protein.

1. The structure of a tetrafunctional cross-link (compound C) isolated from borohydride-reduced cow skin collagen has been determined by the combined use of mass spectrometry and (1)H and (13)C n.m.r. spectroscopy. 2. A new technique, of potentially wide applicability, is described for the mass-spectrometric determination of functional groups. 3. Detailed study of protonation shifts in the (13)C spectrum has allowed an unambiguous assignment of the exact structural relationship of the component parts of the molecule. 4. New interpretations of existing data on this molecule are given.

The detailed mass-spectrometric evidence for our original findings [Magnusson et al. (1974) FEBS Lett. 44, 189-193] of ten gamma-carboxyglutamic acid residues in the N-terminal calcium-binding polypeptide of prothrombin is presented. The identification and sequence location of gamma-carboxyglutamic acid was made by electron-impact and field-desorption studies on acetyl permethyl peptide derivatives, and on the free amino acid. Details of the derivatives formed, and how this new amino acid may be easily recognized and sequenced from the mass spectrum, are given as a basis for future work.

Peptides (residues 1-42) (bovine prothrombin numbering) from bovine Factor X1 and X2 have been separately purified and digested before mass-spectrometric sequence assignment and identification of gamma-carboxyglutamic acid. N-terminal sequence was found to be identical, and 12 residues of gamma-carboxyglutamic acid were unambiguously identified. The new data give conclusive evidence for the N-terminal primary structure of bovine Factor X and extend present knowledge to show (i) unambiguous assignment of gamma-carboxyglutamic acid residues, including a previously unreported residue of gamma-carboxyglutamic acid at position 40 in both Factor X1 and X2, (ii) the physical difference between Factors X1 and X2 is not due to either different amino acid sequences or different gamma-carboxyglutamic acid contents of the N-terminal 42 residues.

The minimal effective concentration of the pituitary insulin secretagogue beta-cell-tropin (beta-CT) on the in vitro perfused pancreas was established and the effects of various modifications of the peptide on its potency were tested: iodination with 127I and acetylation reduced the insulin-releasing activity of beta-cell-tropin, and the C-terminal fragments beta-CT-(2-18), beta-CT-(3-18) and beta-CT-(6-18) were all less potent than the intact molecule; beta-CT-(1-6) was not active and did not inhibit beta-CT-induced insulin release.

An elastase digest of a protein of unknown structure, dihydrofolate reductase, was studied by mass spectrometry. This soluble digest contained a large number of small peptides in different yields, within the ideal molecular-weight range (200-1200) for mixture-analysis mass spectrometry. Sequences of the major component peptides in the digest are reported.

The first detailed results of the application of a low-resolution mixture analysis approach to the sequence analysis of an enzyme, ribitol dehydrogenase, are given. Examples of the interpretation of the spectra of peptide mixtures derived from this protein are described. Evidence for new fragmentation patterns observed is reported, together with an explanation of the generation of ambiguous sequences by use of a low-specificity enzyme, thermolysin. The overall sequencing strategy evolved is assessed.

Several peptides were isolated from the protein silk fibroin of Bombyx mori by means of ion-exchange chromatography of a chymotryptic digest. The sequences of three of the peptides, Gly-Ala-Gly-Tyr, Gly-Val-Gly-Tyr and Gly-Ala-Gly-Ala-Gly-Ala-Gly-Tyr, were known from previous chemical work, but the sequence of the fourth, Gly-Ala-Gly-Val-Gly-Ala-Gly-Tyr, was previously only partially known. The necessary volatility for mass-spectrometric examination of the peptides was achieved by permethylation of the N-acetyl-peptide methyl ester derivatives. From the mass spectra it was possible to confirm the known sequences and to establish that of the partially known one. In one instance it was possible to deduce from the same mass spectrum the sequence of a main peptide component and that of a small amount of contaminating peptide. These results demonstrate for the first time the use of mass spectrometry in the determination of the amino acid sequences in peptides from a protein hydrolysate.

Micro-quantities of protein-derived peptides have been converted into N-acetylated permethyl derivatives, and their sequences determined by low-resolution mass spectrometry without prior knowledge of their amino acid compositions or lengths. A new strategy is suggested for the mass spectrometric sequencing of oligopeptides or proteins, involving gel filtration of protein hydrolysates and subsequent sequence analysis of peptide mixtures. Finally, results are given that demonstrate for the first time the use of mass spectrometry for the analysis of a protein-derived peptide mixture, again without prior knowledge of the protein or components within the mixture.