Mass spectrometry in protein structure analysis

Abstract: Mass spectrometry is an important analytical tool in biological and biochemical research. The speed, accuracy and sensitivity is unmatched by conventional analytical techniques. Identification of proteins and characterisation of their primary structure is a rapidly growing field in the post-genomic era, where matrix-assisted laser desorption/ionisation time-of-flight peptide mass fingerprinting combined with electrospray tandem mass spectrometry is well suited to solve the questions. This thesis deals with the strength and diversity of applying mass spectrometry to biochemical projects. The study covers sample preparation, protein identification, characterisation of post-translational modifications and mechanistic aspects of gas-phase cleavage of protonated peptides. The characteristics of a quadrupole - time-of-flight tandem mass spectrometer, used throughout this study, were investigated in a project where the thyroid hormone receptor ligand binding domain (TR-LBD) was analysed. Carboxymethylation of the TR-LBD in its folded conformation and identification of the alkylated Cys residues allowed estimates of tertiary structure relationships within the protein. In the tryptic digest, the peptides were mass measured with an accuracy of 2 ppm, and product ions generated by tandem mass spectrometry were measured to an accuracy of 20 ppm when internal calibrants were used. Proline-rich proteins (PRPs) are predominant in saliva and nano-electrospray mass spectrometry was employed to identify their degradation products after treatment with Streptococcus and Actinomyces microorganisms. Several of the peptides generated had a Cterminal Pro-Gln bacteria binding sequence, and release of a pentapeptide with innate immunity properties was suggested. In another study it was shown that PRP-1 and its Cterminally truncated form PRP-3 are present in two forms in human saliva, one of which is 0hexuronidated at Ser 17 in the biologically active N-terminal segment. Nano-electrospray tandem mass spectrometry of PRP-1 tryptic peptides was carried out to determine the 0hexuronidation. It was then also found that peptides containing the PQGPPQQGG consensus repeat reveal a facile cleavage of the Gln 2-Gly 3 peptide bond in the gas-phase upon collision-induced dissociation. The data indicate involvement of the G1n side-chain in the fragmentation mechanism. Phosphorylation is a common post-translational modification of proteins but only known in a few prohormone precursors. Using nano-electrospray tandem mass spectrometry, a phosphorylated form of porcine peptide YY was now identified and the modification was localised to Ser 13. In-gel digestion of gel-separated proteins has many advantages in terms of sensitivity and high-throughput for protein identification. However, the coverage of the primary structure by mass spectrometric analysis for detection of post-translational modifications is generally poor. An alternative method was developed that uses electroblotting of the gel-separated proteins to polyvinylidene difluoride membranes followed by protein extraction and digestion in solution. Compared to in-gel digestion, this procedure results in improved sequence coverage (by approximately 30%) and it was applied to identify protein methylation and acetylation sites. DNasel affinity chromatography in combination with nano-electrospray tandem mass spectrometry identified a specific subset of actin-associated heterogeneous nuclear ribonucleoproteins (hnRNP) from rat liver pre-messenger RNP particles. The proteins were identified as being a novel hnRNP A2 isoform, three hnRNP A3 forms and a novel A/B type of hnRNP designated DBP-40. DBP-40 binds directly to actin in vivo, and it is also capable of individually binding RNA as well as hnRNP A2 and A3 in the presence of actin.