Immuno-Mass Spectrometry Workflow for Quantification of Serum α-Fetoprotein Using Antibody-Immobilized Magnetic Beads and Modified Eluents.

Immuno-mass spectrometry (MS) is a powerful method for the quantitative analysis of low-abundance proteins in biological specimens. In these procedures, collecting specifically and efficiently the target protein antigens from the antigen-antibody complex generated on the surface of nanocarrier beads is crucial and can be performed by hydrolyzing the proteins directly on the beads or after elution. Herein, we optimized the conditions of the immunoaffinity purification via elution using serum α-fetoprotein (AFP) as a model and its specific antibody immobilized covalently on magnetic beads. Antibody-coated beads were incubated with human serum spiked with standard AFP for antigen-antibody reaction. AFP was then eluted from the beads using various eluents, including organic solvents, to optimize the elution conditions. After proteolytically hydrolyzing the eluted protein, stable isotope-labeled standard peptides were added to the hydrolysate to quantify the eluted AFP via liquid chromatography-tandem MS. Using an optimized workflow for quantitative analysis afforded a correlation between the amount of spiked AFP and heavy to light ratios calculated based on peptide ion peak areas, from which an endogenous AFP concentration of 2.3±0.6 ng/mL was determined in normal serum; this is consistent with previous reports using radioimmunoassay methods. The present immuno-MS workflow could apply to the detection and quantitation of other low-abundance biofluid biomarkers.

Structural insights of post-translational modification sites in the proteome of Thermus thermophilus.

Phosphorylation and acetylation are the most prevalent post-translational modifications (PTMs) detected in not only eukaryotes but also bacteria. We performed phosphoproteome and acetylome analyses of proteins from an extremely thermophilic eubacterium Thermus thermophilus HB8, and identified numerous phosphorylation and acetylation sites. To facilitate the elucidation of the structural aspects of these PTM events, we mapped the PTM sites on the known tertiary structures for the respective proteins and their homologs. Wu et al. (Mol Cell Proteomics 12:2701-2713, 2013) recently reported phosphoproteome analysis of proteins from T. thermophilus HB27. Therefore, we assessed the structural characteristics of these phosphorylation and acetylation sites on the tertiary structures of the identified proteins or their homologs. Our study revealed that many of the identified phosphosites are in close proximity to bound ligands, i.e., the numbers of 'nearby' and 'peripheral' phosphorylation sites represent 56 % (48/86 sites) of total identified phosphorylation sites. In addition, approximately 60 % of all phosphosites exhibited <10 % accessible surface area of their side chains, suggesting some structural rearrangement is required for phosphoryl transfer by kinases. Our findings also indicate that phosphorylation of a residue occurs more frequently at a flexible region of the protein, whereas lysine acetylation occurs more frequently in an ordered structure.

Close proximity of phosphorylation sites to ligand in the phosphoproteome of the extreme thermophile Thermus thermophilus HB8.

We performed phosphoproteome analysis of proteins from the extremely thermophilic Gram-negative eubacterium Thermus thermophilus HB8 using gel-free mass spectrometric method. We identified 52 phosphopeptides from 48 proteins and determined 46 phosphorylation sites: 30 on serine, 12 on threonine, and 4 on tyrosine. The identified phosphoproteins are known to be involved in a wide variety of cellular processes. To help elucidate the functional roles of these phosphorylation events, we mapped the phosphorylation sites on the known tertiary structures of the respective proteins. In all, we succeeded in mapping 46 sites (approximately 88%) on the corresponding structures. Most of the phosphorylation sites were found to be located on loops and terminal regions of the secondary structures. Surprisingly, 28 of these sites were situated at or near the active site of the enzyme. In particular, 18 sites were within 4 Å of the ligand, including substrate or cofactor. Such structural locations suggest direct effects of the phosphorylation on the binding of ligand in addition to inducing a conformational change. Interestingly, 19 of these 28 phosphorylation sites were situated near the phosphate moiety of a substrate or cofactor. In oligomeric proteins, 5 phosphorylation sites were found at the subunit interface. Based on these results, we propose a regulatory mechanism that involves Ser/Thr/Tyr phosphorylation in T. thermophilus HB8.

A novel single-stranded DNA-specific 3'-5' exonuclease, Thermus thermophilus exonuclease I, is involved in several DNA repair pathways.

Single-stranded DNA (ssDNA)-specific exonucleases (ssExos) are expected to be involved in a variety of DNA repair pathways corresponding to their cleavage polarities; however, the relationship between the cleavage polarity and the respective DNA repair pathways is only partially understood. To understand the cellular function of ssExos in DNA repair better, genes encoding ssExos were disrupted in Thermus thermophilus HB8 that seems to have only a single set of 5'-3' and 3'-5' ssExos unlike other model organisms. Disruption of the tthb178 gene, which was expected to encode a 3'-5' ssExo, resulted in significant increase in the sensitivity to H(2)O(2) and frequency of the spontaneous mutation rate, but scarcely affected the sensitivity to ultraviolet (UV) irradiation. In contrast, disruption of the recJ gene, which encodes a 5'-3' ssExo, showed little effect on the sensitivity to H(2)O(2), but caused increased sensitivity to UV irradiation. In vitro characterization revealed that TTHB178 possessed 3'-5' ssExo activity that degraded ssDNAs containing deaminated and methylated bases, but not those containing oxidized bases or abasic sites. Consequently, we concluded that TTHB178 is a novel 3'-5' ssExo that functions in various DNA repair systems in cooperation with or independently of RecJ. We named TTHB178 as T. thermophilus exonuclease I.

Analysis of a nuclease activity of catalytic domain of Thermus thermophilus MutS2 by high-accuracy mass spectrometry.

Electrospray ionization with Fourier-transform ion cyclotron resonance mass spectrometry (ESI-FT ICR MS) is a powerful tool for analyzing the precise structural features of biopolymers, including oligonucleotides. Here, we described the detailed characterization of a newly discovered nuclease activity of the C-terminal domain of Thermus thermophilus MutS2 (ttMutS2). Using this method, the length, nucleotide content and nature of the 5'- and 3'-termini of the product oligonucleotides were accurately identified. It is revealed that the C-terminal domain of ttMutS2 incised the phosphate backbone of oligodeoxynucleotides non-sequence-specifically at the 3' side of the phosphates. The simultaneous identification of the innumerable fragments was achieved by the extremely high-accuracy of ESI-FT ICR MS.