Protein-sequence polymorphisms and post-translational modifications in proteins from human saliva using top-down Fourier-transform ion cyclotron resonance mass spectrometry

Abstract

Single nucleotide polymorphisms (SNPs) can result in protein sequence polymorphisms (PSPs) when codon translations are altered. Both top-down and bottom-up proteomics strategies can identify PSPs, but only if databases and software are used with this in mind. A 14319 Da protein from human saliva was characterized using the top-down approach on a hybrid linear ion-trap Fourier-transform ion cyclotron resonance mass spectrometer equipped for both collisionally-activated (CAD) and electron-capture (ECD) dissociation. Sequence tags identified the protein as Cystatin SN, and defined the N-terminal signal peptide cleavage site, as well as two disulfide bonds, in agreement with previous studies. The mass of the intact protein (< 5 ppm error) deviated from that calculated from the published gene sequence by 16.031 Da, and, based on CAD and ECD fragment ion assignments, it was concluded that the isoform of the protein analyzed carried a PSP at residue 11 such that the Pro translated from the genome was in fact Leu/Ile. An independently determined SNP (rs2070856) subsequently confirmed the genetic basis of the mass spectral interpretation and defined the residue as Leu. In another example, the PRP3 protein with mass ∼10,999 Da was found to be an isomeric/ isobaric mixture of the reported sequence with PSPs D4N or D50N (rs1049112). Both CAD and ECD datasets support two phosphorylation sites at residues Ser8 and Ser22, rather than Ser17. In the

Translation Control of Swarming Proficiency in Bacillus subtilis by 5-Amino-pentanolylated Elongation Factor P

“…High-resolution Top-Down Mass Spectrometry-Individual native and recombinant B. subtilis EF-P was initially analyzed as described before by tandem LC-MSϩ using a triple-quadrupole mass spectrometer (API IIIϩ, Applied Biosystems) prior to being introduced to Fourier transform-ICR by direct infusion nanospray (6,26,27). Envelopes of multiply charged ions were measured for each sample on a hybrid linear ion-trap/ Fourier transform-ICR mass spectrometer (7T, LTQ FT Ultra, Thermo Scientific) operated with a standard (up to m/z 2000) or extended mass range (up to m/z 4000).…”

Post-translational Modifications of Integral Membrane Proteins Resolved by Top-down Fourier Transform Mass Spectrometry with Collisionally Activated Dissociation

“…Such an observation is usually an indication that the complete sequence is covered and correct, but in this case, where the difference between precursor and sequence is 274 Da due to the presence of the bound inhibitor, the data must be examined further to explain the discrepancy. The observation of overlapping b-and y-ions could arise in two ways by either neutral loss, whereby some product ions that carried the modification lost it such that their measured mass corresponded to the unmodified ion, or the presence of mixed populations with modifications at both Cys residues (24). The neutral loss problem can be solved by relying solely upon product ions that carry the modification.…”

Protein Identification Using Top-Down Spectra

“…various proteolytic forms of the same protein or various protein isoforms resulting from alternative splicing. Top-down mass spectrometry focuses on analyzing intact proteins and large peptides (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) and has advantages in localizing multiple post-translational modifications (PTMs) 1 in a coordinated fashion (e.g. combinatorial PTM code) and identifying multiple protein species (e.g.…”