Incresing the system peak capacity of LC-MS/MS work flows for qualitative and quantitative protein profiling by incorporating ion mobility separations

Comunicaciones a congreso

Further development of high-sensitivity targeted MRM Assays for peptide and protein quantification

Comunicaciones a congreso

The Propeptide Binding Site of the Bovine γ-Glutamyl Carboxylase

gamma-Glutamyl carboxylase is an integral membrane protein required for the posttranslational modification of vitamin K-dependent proteins. The main recognition between the enzyme and its substrates is through an 18-amino acid propeptide. It has been reported that this binding site resides in the amino-terminal third of the gamma-glutamyl carboxylase molecule (Yamada, M., Kuliopulos, A., Nelson, N. P., Roth, D. A., Furie, B., Furie, B. C., and Walsh, C. T. (1995) Biochemistry 34, 481-489). In contrast, we found the binding site in the carboxyl half of the gamma-glutamyl carboxylase. We show that the carboxylase may be cleaved by trypsin into an amino-terminal 30-kDa and a carboxyl-terminal 60-kDa fragment joined by a disulfide bond(s), and the propeptide binds to the 60-kDa fragment. The sequence of the amino terminus of the 60-kDa fragment reveals that the primary trypsin-sensitive sites are at residues 349 and 351. Furthermore, the tryptic fragment that cross-links to the propeptide also reacts with an antibody specific to the carboxyl portion of the gamma-glutamyl carboxylase. In addition, cyanogen bromide cleavage of bovine gamma-glutamyl carboxylase cross-linked to the peptide comprising residues TVFLDHENANKILNRPKRY of human factor IX yields a cross-linked fragment of 16 kDa from the carboxyl half of the molecule, the amino-terminal sequence of which begins at residue 438. Thus, the propeptide binding site lies carboxyl-terminal to residue 438 and is predicted to be in the lumen of the endoplasmic reticulum

Profiling invasive Plasmodium falciparum merozoites using an integrated omics approach

The symptoms of malaria are brought about by blood-stage parasites, which are established when merozoites invade human erythrocytes. Our understanding of the molecular events that underpin erythrocyte invasion remains hampered by the short-period of time that merozoites are invasive. To address this challenge, a Plasmodium falciparum gamma-irradiated long-lived merozoite (LLM) line was developed and investigated. Purified LLMs invaded erythrocytes by an increase of 10–300 fold compared to wild-type (WT) merozoites. Using an integrated omics approach, we investigated the basis for the phenotypic difference. Only a few single nucleotide polymorphisms within the P. falciparum genome were identified and only marginal differences were observed in the merozoite transcriptomes. By contrast, using label-free quantitative mass-spectrometry, a significant change in protein abundance was noted, of which 200 were proteins of unknown function. We determined the relative molar abundance of over 1100 proteins in LLMs and further characterized the major merozoite surface protein complex. A unique processed MSP1 intermediate was identified in LLM but not observed in WT suggesting that delayed processing may be important for the observed phenotype. This integrated approach has demonstrated the significant role of the merozoite proteome during erythrocyte invasion, while identifying numerous unknown proteins likely to be involved in invasion

Protein markers for insulin-producing beta cells with higher glucose sensitivity

Background and Methodology: Pancreatic 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 Findings: All 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. Conclusions: Quantification 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

The use of proteome similarity for the qualitative and quantitative profiling of reperfused myocardium.

International audienceAn LC-MS-based approach is presented for the identification and quantification of proteins from unsequenced organisms. The method relies on the preservation of homology across species and the similarity in detection characteristics of proteomes in general. Species related proteomes share similarity that progresses from the amino acid frequency distribution to the complete amino sequence of matured proteins. Moreover, the comparative analysis between theoretical and experimental proteome distributions can be used as a measure for the correctness of detection and identification obtained through LC-MS-based schemes. Presented are means to the identification and quantification of rabbit myocardium proteins, immediately after inducing cardiac arrest, using a data-independent LC-MS acquisition strategy. The employed method of acquisition affords accurate mass information on both the precursor and associated product ions, whilst preserving and recording the intensities of the ions. The latter facilitates label-free quantification. The experimental ion density observations obtained for the rabbit sub proteome were found to share great similarity with five other mammalian samples, including human heart, human breast tissue, human plasma, rat liver and a mouse cell line. Redundant, species-homologues peptide identifications from other mammalian organisms were used for initial protein identification, which were complemented with peptide identifications of translated gene sequences. The feasibility and accuracy of label-free quantification of the identified peptides and proteins utilizing above mentioned strategy is demonstrated for selected cardiac rabbit proteins

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