Strategy for surveying the proteome using affinity proteomics and mass spectrometry

Abstract

Radiation-induced gliomas represent a relatively rare but well-characterized entity in the neuro-oncologic literature. Extensive retrospective cohort data in pediatric populations after therapeutic intracranial radiation show a clearly increased risk in glioma incidence that is both patient age- and radiation dose/volume-dependent. Data in adults are more limited but show heightened risk in certain groups exposed to radiation. In both populations, there is no evidence linking increased risk associated with routine exposure to diagnostic radiation. At the molecular level, recent studies have found distinct genetic differences between radiation-induced gliomas and their spontaneously-occurring counterparts. Clinically, there is understandable reluctance on the part of clinicians to re-treat patients due to concern for cumulative neurotoxicity. However, available data suggest that aggressive intervention can lead to improved outcomes in patients with radiation-induced gliomas.

Epitope‐specificity of recombinant antibodies reveals promiscuous peptide‐binding properties

“…However, in our case, this was a deliberate motif strategy based on the fact that the GPS set-up was designed to target tryptic peptides. [21][22][23] It could be anticipated that the same phenomena would be observed using proteases favoring different cutting sites, although this remains to be experimentally verified. The impact of adding extra residues to the C-terminal end of the peptides also remains to be directly evaluated.…”

“…The set of tryptic peptides bound by each individual CIMS antibody was thus detected and identified using tandem-mass spectrometry. The results showed that peptides of a median length of 10 amino acids (range [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] and with a median pI of 6.5 (range 3.8-12) were captured in a clone-dependent manner (Table I). Noteworthy, as illustrated for the CIMS-17 sister pair, the affinity (K D ) for an experimentally verified captured yeast peptide was found to be in the same range as that observed for the original selection peptide (Table I).…”

“…In order to make sure that the phenomenon of degenerate peptide-binding specificity was biologically relevant and not a bias introduced by the library design, we examined the reactivity pattern of two conventional antibodies raised in rabbits against similar tryptic peptide motifs. 33 Of note, these antibodies are used within a set-up similar to our GPS technology platform, [21][22][23] denoted Triple X proteomics (TXP). 33 Most interestingly, we found three semi-conserved resides (P4-x-P2-P1 or P4-P3-x-P1) to be essential for establishing the peptide-binding specificity also for these antibodies, while a higher degree of wobbling was allowed in the other positions (Supporting Information Fig.…”

“…[21][22][23] In this set-up, we analyze proteomes generated by tryptic digestion using human recombinant single-chain Fv (scFv) antibodies directed against short binding motifs. Each motif, 4-6 amino acids long, is shared among several proteins (up to several hundred), enabling each antibody to target numerous proteins in a specific independent manner.…”

Proteomics of spermatogenesis: from protein lists to understanding the regulation of male fertility and infertility

“…However, LC-MS cannot match proteins identified by software with their Mr and pI, and another major limitation is that this technique is not suitable for quantitative analysis. 59,65 To overcome the shortcomings of 2DE and LC as individual techniques, this study 21,30,32 combined 2DE and LC to explore protein changes in cells and tissues. In mouse testis, we used 2DE to construct an overall proteome profile and comparative proteome profiles of different stages of spermatogenesis, which revealed that many proteins appeared as two or more spots.…”