Search

Filter-aided sample preparation is a proteomic technique for the preparation and on column proteolysis of proteins. Recently an enhanced FASP protocol was developed that uses deoxycholic acid (DCA) and that reportedly enhances trypsin proteolysis, resulting in increases cytosolic and membrane protein representation. FASP and eFASP were re-evaluated by ultra-high-performance liquid chromatography coupled to a quadrupole mass filter Orbitrap analyzer (Q Exactive). Although there was no difference in trypsin activity, 14,099 and 13,414 peptides, describing 1723 and 1793 protein groups, from Escherichia coli K12 were identified using FASP and eFASP, respectively. Characterization of the physicochemical properties of identified peptides showed no significant differences other than eFASP extracting slightly more basic peptides. At the protein level, both methods extracted essentially the same number of hydrophobic transmembrane helix-containing proteins as well as proteins associated with the cytoplasm or the cytoplasmic and outer membranes. By employing state-of-the-art LC-MS/MS shot gun proteomics, our results indicate that FASP and eFASP showed no significant differences at the protein level. However, because of the slight differences in selectivity at the physicochemical level of peptides, these methods can be seen to be somewhat complementary for analyses of complex peptide mixtures.

Background: The central role of proteins in diseases has made them increasingly attractive as therapeutic targets and indicators of cellular processes. Protein microarrays are emerging as an important means of characterising protein activity. Their accurate downstream analysis to produce biologically significant conclusions is largely dependent on proper pre-processing of extracted signal intensities. However, existing computational tools are not specifically tailored to the nature of these data and lack unanimity. Results: Here, we present the single-channel Protein Microarray Analysis Pipeline, a tailored computational tool for analysis of single-channel protein microarrays enabling biomarker identification, implemented in R, and as an interactive web application. We compared four existing background correction and normalization methods as well as three array filtering techniques, applied to four real datasets with two microarray designs, extracted using two software programs. The normexp, cyclic loess, and array weighting methods were most effective for background correction, normalization, and filtering respectively. Conclusions: Thus, here we provided a versatile and effective pre-processing and differential analysis workflow for single-channel protein microarray data in form of an R script and web application (https://metaomics.uct.ac.za/shinyapps/Pro-MAP/.) for those not well versed in the R programming language. [ABSTRACT FROM AUTHOR]

Ser/Thr/Tyr protein phosphorylation plays a critical role in regulating mycobacterial growth and development. Understanding the mechanistic link between protein phosphorylation signaling network and mycobacterial growth rate requires a global view of the phosphorylation events taking place at a given time under defined conditions. In the present study we employed a phosphopeptide enrichment and high throughput mass spectrometry-based strategy to investigate and qualitatively compare the phosphoproteome of two mycobacterial model organisms: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG. Cells were harvested during exponential phase and our analysis detected a total of 185 phospho-sites in M. smegmatis, of which 106 were confidently localized [localization probability (LP) = 0.75; PEP = 0.01]. By contrast, in M. bovis BCG the phosphoproteome comprised 442 phospho-sites, of which 289 were confidently localized. The percentage distribution of Ser/Thr/Tyr phosphorylation was 39.47, 57.02, and 3.51% for M. smegmatis and 35, 61.6, and 3.1% for M. bovis BCG. Moreover, our study identified a number of conserved Ser/Thr phosphorylated sites and conserved Tyr phosphorylated sites across different mycobacterial species. Overall a qualitative comparison of the fast and slow growing mycobacteria suggests that the phosphoproteome of M. smegmatis is a simpler version of that of M. bovis BCG. In particular, M. bovis BCG exponential cells exhibited a much more complex and sophisticated protein phosphorylation network regulating important cellular cycle events such as cell wall biosynthesis, elongation, cell division including immediately response to stress. The differences in the two phosphoproteomes are discussed in light of different mycobacterial growth rates. [ABSTRACT FROM AUTHOR]