Next generation proteomics allows precise quantitative analysis of bacterial secretion system and centrosome architecture

Date&Time: 18 October 2019 - h. 2:00 pm
Location: Via Sommarive 9 - Polo Ferrari 2 (Povo, TN) - Room B103

Speaker

• Alexander Schmidt

Head of the Proteomics Core Facility, Biozentrum, University of Basel, Basel, Switzerland

Abstract:

Quantitative biology relies on data sets in which the same group of proteins is consistently identified and precisely quantified across multiple samples, a requirement that is only partially achieved by current shotgun proteomics approaches. The development of targeted LC-MS/MS methods ideally complements the discovery capabilities of shotgun strategies by its unique potential for reliable quantification of specific proteins within complex mixtures. The recent development of fast scanning high-resolution/accurate-mass LC-MS platforms in combination with cheap, high-throughput synthesis of heavy reference peptides have dramatically improved the sensitivity and precision of quantitative targeted LC-MS analysis. Using this parallel reaction monitoring approach, it is now possible to absolutely quantify selected proteins of interest down to a few hundred copies per cell directly from total cell lysates requiring only minute amounts of samples. Thus, targeted LC-MS represents a cheap and straightforward alternative to common antibody-based approaches, like western blot or ELISA, in particular if suited antibodies are not available. To demonstrate the power of this workflow, we applied it to determine the absolute abundance levels of bacterial Type VI (T6SS) secretion system key components in Vibrio cholerae, Pseudomonas aeruginosa and Acinetobacter baylyi to gain insights into the mechanisms regulating T6SS assembly[1]. Moreover, we employed it to monitor changes in the cellular abundance of centriole duplication key factors to study centriole biogenesis and to precisely quantify[2] and localize methyl histone marks directly from human cell lysates. As the strategy is relatively fast, inexpensive and applicable to any sample type, we expect that targeted LC-MS will become a cornerstone methodology of quantitative biology. [1] L. Lin, E. Lezan, A. Schmidt, and M. Basler, “Abundance of bacterial Type VI secretion system components measured by targeted proteomics.,” Nat Commun, vol. 10, no. 1, p. 2584, Jun. 2019.[2] C. Arquint, F. Cubizolles, A. Morand, A. Schmidt, and E. A. Nigg, “The SKP1-Cullin-F-box E3 ligase βTrCP and CDK2 cooperate to control STIL abundance and centriole number.,” Open Biol, vol. 8, no. 2, p. 170253, Feb. 2018.