Andreas P. Jonsson

Gln-Gly cleavage: correlation between collision-induced dissociation and biological degradation

Tryptic digestion of the 150-residue human acidic salivary proline-rich protein 1 (PRP-1) generated eight peptides, two of which corresponded to the N-terminal 30-residue segment. In each of the other six tryptic peptides, a consensus repeat with the structure PQGPPQQGG was present. A facile Gln-Gly cleavage between the second and the third residues of the repeat was observed during collision-induced dissociation experiments. We postulate possible mechanisms to account for this reactivity, involving attack on the peptidyl carbonyl group by the Gln sidechain. Significantly, the Gln-Gly cleavage has been shown to be biologically important in the bacterial degradation of PRPs in saliva, generating bacteria-binding Pro-Gln C-termini. We suggest a link between the gas-phase chemistry and the biochemical degradation of these molecules.

Possible Release of an ArgGlyArgProGln Pentapeptide with Innate Immunity Properties from Acidic Proline-Rich Proteins by Proteolytic Activity in Commensal Streptococcus and Actinomyces Species

ABSTRACT This study suggests degradation of salivary acidic proline-rich proteins (PRPs) into potential innate-immunity-like peptides by oralStreptococcus and Actinomyces species. PRP degradation paralleled cleavage of Pro-containing substrates. PRP degradation by S. gordonii strain SK12 instantly released a Pyr1-Pro104Pro105 and a Gly111-Pro149Gln150 peptide together with a presumed Arg106Gly107Arg108Pro109Gln110pentapeptide. The synthetic Arg106Gly107Arg108Pro109Gln110peptide desorbed bound bacteria and counteracted sucrose-induced decrease of dental plaque pH in vitro.

Ser13-phosphorylated PYY from porcine intestine with a potent biological activity

We have isolated a posttranslationally modified form of peptide YY (PYY) from porcine intestine and shown by MALDI‐TOF and electrospray tandem mass spectrometry that it is phosphorylated at Ser13. Phospho‐PYY exhibits high affinity for binding to neuropeptide Y (NPY) receptors Y1, Y2 and Y5. The IC50 values with the Y1, Y2, and Y5 receptor subtypes were for NPY 2.4, 3.1, and 3.3 nM, for PYY 2.3, 0.94, and 3.2 nM, and for phospho‐PYY 4.6, 2.2, and 5.5 nM, respectively. Phospho‐PYY potently inhibits forskolin‐stimulated cAMP accumulation in SK‐N‐MC cells with an IC50 value of 0.5 nM compared to 0.15 nM for non‐phosphorylated PYY. The finding of phosphorylation of PYY is unusual among hormonal peptides, and emphasizes the importance of direct protein analysis of gene products.

A novel Ser O-glucuronidation in acidic proline-rich proteins identified by tandem mass spectrometry

Human acidic proline‐rich salivary protein PRP‐1 and its C‐terminally truncated form PRP‐3 were analyzed by electrospray tandem mass spectrometry. Post‐translational modifications were detected and characterized. A pyroglutamic acid residue was demonstrated at the N‐terminus, Ser‐8 and Ser‐22 were shown to be phosphorylated and an O‐linked glucuronic acid conjugation was identified. The latter modification was located to Ser‐17 and found to be present in approximately 40% of the polypeptides.

Structural analysis of the thyroid hormone receptor ligand binding domain: studies using a quadrupole time-of-flight tandem mass spectrometer

The overall architecture of the ligand binding domain (LBD) of members of the nuclear receptor superfamily are similar. There are now standard procedures to express and purify these proteins. A rapid and sensitive method for the structural analysis of these proteins is nano-electrospray tandem mass spectrometry. In the present study we have analysed the LBD of the human thyroid hormone receptor-beta-1 (TR-beta) by quadrupole time-of-flight tandem mass spectrometry. The intact protein was analysed in a carboxymethylated form in an attempt to identify which cysteine residues are located on the surface. The protein molecular weight (31 652.5 Da) was determined with an accuracy of +/-1 Da, while masses of tryptic fragments were determined with an accuracy of at least 75 ppm. The sequence coverage of the tryptic peptide mass map was 93.2 %. Tryptic peptides were subjected to collision-induced dissociation (CID) and the resulting product ions were mass measured with an accuracy of about 100 ppm. When accurate mass measurements were made with internal calibration, mass accuracies were improved to +/-2 ppm in mass spectra, and +/-20 ppm in CID spectra. From these data it was possible to determine the presence of post-translational modifications, locate the sites of carboxymethylation and, in addition, confirm the amino acid sequence of the expressed protein. To the best of our knowledge, this is the first characterisation of the TR-LBD-beta at the protein level.

Gas phase conformation can have an influence on peptide fragmentation

In the post‐genomic era, mass spectrometry is destined to fulfil a central role in biomedical research, and it is in the area of protein identification that mass spectrometry is now most rapidly expanding. An important identification method is to subject a protein to proteolysis and determine the resulting peptide masses and/or primary structure. From such determinations proteins can be identified. Tandem mass spectrometry (MS/MS) is used to determine primary structure and, for high‐throughput identification, computer‐based automated strategies are a prerequisite. Computer programs are available for such identifications, where simulated MS/MS spectra of amino acid sequences within a database are generated and compared to experimental spectra. Such algorithms take into account empirical rules for peptide fragmentation, rather than specific gas‐phase ion chemistry. For example, fragmentation of each peptide bond is usually considered to be equally facile. In reality, this is not the case. Gas‐phase ion chemistry bears an important role in determining the abundance of fragment ions in MS/MS spectra. In this communication, the gas‐phase ion chemistry responsible for the facile cleavage between Gln and Gly residues is investigated, particularly in relation to Proline Rich Protein‐1.

Characterization of new medium-chain alcohol dehydrogenases adds resolution to duplications of the class I/III and the sub-class I genes

Four additional variants of alcohol and aldehyde dehydrogenases have been purified and functionally characterized, and their primary structures have been determined. The results allow conclusions about the structural and evolutionary relationships within the large family of MDR alcohol dehydrogenases from characterizations of the pigeon (Columba livia) and dogfish (Scyliorhinus canicula) major liver alcohol dehydrogenases. The pigeon enzyme turns out to be of class I type and the dogfish enzyme of class III type. This result gives a third type of evidence, based on purifications and enzyme characterization in lower vertebrates, that the classical liver alcohol dehydrogenase originated by a gene duplication early in the evolution of vertebrates. It is discernable as the major liver form at about the level in-between cartilaginous and osseous fish. The results also show early divergence within the avian orders. Structures were determined by Edman degradations, making it appropriate to acknowledge the methodological contributions of Pehr Edman during the 65 years since his thesis at Karolinska Institutet, where also the present analyses were performed.

Gas-Phase Conformation Can Have an Influence on Peptide Fragmentation

In the post-genomic era, mass spectrometry is destined to fulfil a central role in biomedical research, and it is in the area of protein identification that mass spectrometry is now most rapidly expanding. An important identification method is to subject a protein to proteolysis and determine the resulting peptide masses and/or primary structure. From such determinations proteins can be identified. Tandem mass spectrometry (MS/MS) is used to determine primary structure and, for high-throughput identification, computer-based automated strategies are a prerequisite. Computer programs are available for such identifications, where simulated MS/MS spectra of amino acid sequences within a database are generated and compared to experimental spectra. Such algorithms take into account empirical rules for peptide fragmentation, rather than specific gas-phase ion chemistry. For example, fragmentation of each peptide bond is usually considered to be equally facile. In reality, this is not the case. Gas-phase ion chemistry bears an important role in determining the abundance of fragment ions in MS/MS spectra. In this communication, the gas-phase ion chemistry responsible for the facile cleavage between Gln and Gly residues is investigated, particularly in relation to Proline Rich Protein-1.