Defining intact protein primary structures from saliva: a step toward the human proteome project.

Top-down mass spectrometry has been used to investigate structural diversity within some abundant salivary protein families. In this study, we report the identification of two isoforms of protein II-2 which differed in mass by less than 1 Da, the determination of a sequence for protein IB8a that was best satisfied by including a mutation and a covalent modification in the C-terminal part, and the assignment of a sequence of a previously unreported protein of mass 10433 Da. The final characterization of Peptide P-J was achieved, and the discovery of a truncated form of this peptide was reported. The first sequence assignment was done at low resolution using a hybrid quadrupole time-of-flight instrument to quickly identify and characterize proteins, and data acquisition was switched to Fourier-transform ion cyclotron resonance (FTICR) for proteins that required additional sequence coverage and certainty of assignment. High-resolution and high mass accuracy mass spectrometry on a FTICR-mass spectrometry (MS) instrument combined with electron-capture dissociation (ECD) provided the most informative data sets, with the more frequent presence of "unique" ions that unambiguously define the primary structure. A mixture of predictable and unusual post-translational modifications in the protein sequence precluded the use of shotgun-annotated databases at this stage, requiring manual iterations of sequence refinement in many cases. This led us to propose guidelines for an iterative processing workflow of MS and MSMS data sets that allow researchers to completely assign the identity and the structure of a protein.

Analysis of plant phosphatidylcholines by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) for the quantitative determination of phospholipid (PL) molecular species has been problematic, due primarily to the formation of multiple signals (corresponding to the molecular ion and other adducts) for some classes of PL. For example, analysis of phosphatidylcholine (PC) yielded signals that corresponded to protonated and sodiated molecules in the MALDI spectrum. The resulting spectral overlap among various molecular species (e.g. [PC(16:0/18:2) + Na] and [PC(18:2/18:3)]) made it impossible to ascertain their relative amounts using this technique. Other spectral ambiguities existed among different structural isomers, such as PC(18:1/18:1) and PC(18:0/18:2). We determined that molecular species could be resolved by MALDI-TOFMS by first removing the polar head (e.g. phosphocholine) from the phospholipid to effect production of only the sodiated molecules of the corresponding diacylglycerols (DAGs). Analysis of the resulting spectrum allowed unequivocal determination of the molecular species profile of PC from potato tuber and soybean. Estimation of fatty acid composition based on the molecular species determined by MALDI-TOFMS analysis agreed with that from GC-FID analysis. Post-source decay (PSD) was used to resolve standard isomers of PC (e.g. 18:1/18:1 vs. 18:0/18:2). Our results indicated that PSD is a useful approach for resolving structural isomers of PL molecular species.