The proteome of the recently discovered bacterium Methylocella silvestris has been characterized using three profiling and comparative proteomics approaches. The organism has been grown on two different substrates enabling variations in protein expression to be identified. The results obtained using the experimental approaches have been compared with respect to number of proteins identified, confidence in identification, sequence coverage and agreement of regulated proteins. The sample preparation, instrumental time and sample loading requirements of the differing experiments are compared and discussed. A preliminary screen of the protein regulation results for biological significance has also been performed.

We report automated and time efficient (2 h per sample) profiling of muscle using ultra-performance liquid chromatography (LC) coupled directly with high-definition mass spectrometry (HDMSE). Soluble proteins extracted from rat gastrocnemius (n=10) were digested with trypsin and analysed in duplicate using a 90 min RPLC gradient. Protein identification and label-free quantitation were performed from HDMSE spectra analysed using TransOmics Informatics for Proteomics software. In total 1,514 proteins were identified. Of these, 811 had at least 3 unique peptides and were subsequently used to assess the dynamic range and precision of LC-HDMSE label-free profiling. Proteins analysed by LC-HDMSE encompass the entire complement of glycolytic, beta-oxidation and tricarboxylic acid enzymes. In addition, numerous components of the electron transport chain and protein kinases involved in skeletal muscle regulation were detected. The dynamic range of protein abundances spanned 4 orders of magnitude. The correlation between technical replicates of the 10 biological samples was R2 = 0.9961 ± 0.0036 (95 % CI = 0.9940 – 0.9992) and the technical coefficient of variation averaged 7.3 ± 6.7 % (95 % CI = 6.87 – 7.79 %). This represents the most sophisticated label-free profiling of skeletal muscle to date.

Background and MethodologyPancreatic beta cells show intercellular differences in their metabolic glucose sensitivity and associated activation of insulin production. To identify protein markers for these variations in functional glucose sensitivity, rat beta cell subpopulations were flow-sorted for their level of glucose-induced NAD(P)H and their proteomes were quantified by label-free data independent alternate scanning LC-MS. Beta cell-selective proteins were also identified through comparison with rat brain and liver tissue and with purified islet alpha cells, after geometrical normalization using 6 stably expressed reference proteins.Principal FindingsAll tissues combined, 943 proteins were reliably quantified. In beta cells, 93 out of 467 quantifiable proteins were uniquely detected in this cell type; several other proteins presented a high molar abundance in beta cells. The proteome of the beta cell subpopulation with high metabolic and biosynthetic responsiveness to 7.5 mM glucose was characterized by (i) an on average 50% higher expression of protein biosynthesis regulators such as 40S and 60S ribosomal constituents, NADPH-dependent protein folding factors and translation elongation factors; (ii) 50% higher levels of enzymes involved in glycolysis and in the cytosolic arm of the malate/aspartate-NADH-shuttle. No differences were noticed in mitochondrial enzymes of the Krebs cycle, beta-oxidation or respiratory chain.ConclusionsQuantification of subtle variations in the proteome using alternate scanning LC-MS shows that beta cell metabolic glucose responsiveness is mostly associated with higher levels of glycolytic but not of mitochondrial enzymes.

Butyrate is a critical cancer-preventive element in the colon milieu whose mechanism of action is unclear, but appears to be mediated through inhibition of histone deacetylases (HDACs) and consequent alterations in global protein acetylation. Cytokeratins (CKs) have roles in cytoskeletal function as components of the intermediate filaments (IFs) and this involves CKs in the regulation of tissue homeostasis of high-turnover epithelia such as the colon. We used a 2-D gel/MS analysis to characterise the proteome of IFs, and a novel monitoring-initiated detection and sequencing (MIDAS) approach to identify acetylation sites on principal proteins. We report that CKs are highly acetylated in a colon cancer cell line, with five acetylation sites characterised on CK8 and a further one on CK18. Acetylation of CK8 is responsive to butyrate. HDAC5 is the deacetylase associated with IFs. These data indicate a novel action of butyrate as a cancer preventive agent. Acetylation of CK8 may be associated with IFs stabilisation and thereby provide a candidate mechanism for the appropriate retention or loss of epithelial cells from the flat mucosa.