Dataset Information

A deeper look at carrier proteome effects for single-cell proteomics

ABSTRACT: We probe the carrier proteome effects in single cell proteomics with mixed species TMTpro-labeled samples. We demonstrate that carrier proteomes, while increasing overall identifications, dictate which proteins are identified. We show that quantitative precision and signal intensity are limited at high carrier levels, hindering the recognition of regulated proteins. Guidelines for optimized mass spectrometry acquisition parameters and best practices for fold-change or protein copy number-based comparisons are provided.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human) Escherichia Coli Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Zilu Ye

LAB HEAD: Jesper V. Olsen

PROVIDER: PXD027742 | Pride | 2022-02-24

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20200820_EXPL8_EVO1_ZY_SA_44min_TMT_EY14_100pg_AGC300__1-_1_.pdResult	Other
	20200820_EXPL8_EVO1_ZY_SA_44min_TMT_EY14_100pg_AGC300__1-_2_.pdResult	Other
	20200820_EXPL8_EVO1_ZY_SA_44min_TMT_EY14_100pg_AGC300__1-_3_.pdResult	Other
	20200820_EXPL8_EVO1_ZY_SA_44min_TMT_EY14_100pg_AGC300__1.pdResult	Other
	20200820_EXPL8_EVO1_ZY_SA_44min_TMT_EY14_100pg_AGC300__1.raw	Raw

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Project description:SUMO modification of proteins (sumoylation) is essential for mitotic progression from yeast to humans, but only a limited number of sumoylated proteins with functions in mitosis have been discovered. Vertebrates express three SUMO paralogs, SUMO-1, SUMO-2 and SUMO-3, but only SUMO-2 and -3 are chromosome-associated in mitosis. In this study, we used chromosome spreads to more precisely define the localization of endogenous SUMO-2 and -3 to the centromere as well as the chromosome protein scaffold. Furthermore, we developed methodologies for the immunopurification of endogenous SUMO-2 and -3 modified proteins from cell extracts. Using LC-MS/MS, we analyzed proteins immunopurified from mitotic chromosome fractions and G0 nuclear fractions. We identified 296 putative sumoylated proteins, with 138 being modified specifically in mitosis. We identified proteins known to localize to the centromere and kinetochore and the chromosome protein scaffold, consistent with SUMO-2/3 localization. Furthermore, we demonstrate that utilizing cell synchronization and fractionation allowed for the discovery of low abundance sumoylated proteins that are not detected in large asynchronous analyses. Our results provide a foundation for further characterization of the roles of sumoylation in regulating diverse aspects of chromosome segregation in mitosis. Thermo .raw files were uploaded to the ProHits (Liu et al, 2010) analytical suite and converted to .mzXML format using ReAdW software. Data were searched using X!Tandem (Craig & Beavis, 2004) against human ORFs (RefSeq v45). Search parameters specified a parent MS tolerance of +/- 15ppm, and an MS/MS tolerance of 0.4 Da, with up to two missed cleavages for trypsin. Oxidation of methionine and tryptophan, ubiquitylation of lysine, and alkylation of cysteine (by NEM) were allowed as variable modifications. Statistical validation of the results was performed using Peptide Prophet and Protein Prophet (Keller et al, 2002; Nesvizhskii et al, 2003) as part of the trans-proteomic pipeline. For each search, the Protein Prophet probability at a 1% false discovery rate was used as a cutoff value to generate SAINT-compatible input files. SAINT parameters were as follows: 5000 iterations, low mode Off (0), minFold 1 and normalization (1) (Choi et al, 2011). SAINT cutoff values of 0.75 were used to generate a high-confidence list of protein identifications.

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A deeper look at carrier proteome effects for single-cell proteomics

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