Dataset Information

ChaFRADIC: A novel SCX-based approach for N-terminal Enrichment

ABSTRACT: Step 1: Cell lysis of yeast sphaeroblast (Saccharomyces cerevisiae) and carbamidomethylation. Step 2: Isotopic labeling with dimethylation (light + medium). Step 3: Proteolytic digestion with trypsin (ArgC specificity because of blocked lysines by dimethylation). Step 4: Enrichment via ChaFRADIC method Step 5: Nano-LC-MS/MS measurements Step 6: Data interpretation. Specifications: --ESI spray ionisation o Q Exactive mass spec. --Quadrupole selection, HCD fragmentation and orbitrap detection o Identification + Quantification: PD version 1.3.0039. Search engine: Mascot 2.4. SGD database (Sep 2011), 6717 target sequences. Special nodes: precursor ions quantifier (within a 1 min retention time window), percolator (default settings). Protease: SemiArgC with max. 2 missed cleavage sites. Used modifications: Yeast_N-term_Enriched_Search_Acetylation Variable -- N-terminal Acetylation (42.0105 Da) -- Dimethylation light (28.0313 Da) at Lys -- Dimethylation medium (34.0689 Da) at Lys fixed -- Carbamidomethylation at Cys (57.0214 Da) Quantification -- Peptides with labeled lysine Yeast_N-term_Enriched_Search_Dimethylation Variable -- N-terminal Dimethylation light (28.0313 Da) -- N-terminal Dimethylation medium (34.0689 Da) -- Dimethylation light (28.0313 Da) at Lys -- Dimethylation medium (34.0689 Da) at Lys fixed -- Carbamidomethylation at Cys (57.0214 Da) Quantification -- Peptides with labeled N-terminus -- Peptides with labeled N-terminus and labeled lysine. Mass tolerance: 10 ppm for MS, 0.02 Da for MS/MS. Applied filters: high confidence (FDR <1%), search engine rank 1

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Saskia Venne

PROVIDER: PXD000292 | Pride | 2013-08-09

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	Yeast_N-term_Enriched_Search_Acetylation.msf	Msf
	Yeast_N-term_Enriched_Search_Acetylation.pep.pep.xml	Pepxml
	Yeast_N-term_Enriched_Search_Dimethylation.msf	Msf
	Yeast_N-term_Enriched_Search_Dimethylation.pep.xml	Pepxml
	enriched_fr.1.mzML	Mzml

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Publications

Novel highly sensitive, specific, and straightforward strategy for comprehensive N-terminal proteomics reveals unknown substrates of the mitochondrial peptidase Icp55.

Venne A Saskia AS Vögtle F-Nora FN Meisinger Chris C Sickmann Albert A Zahedi René P RP

Journal of proteome research 20130821 9

We present a novel straightforward method for enrichment of N-terminal peptides, utilizing charge-based fractional diagonal chromatography (ChaFRADIC). Our method is robust, easy to operate, fast, specific, and more sensitive than existing methods, enabling the differential quantitation of 1459 nonredundant N-terminal peptides between two S. cerevisiae samples within 10 h of LC-MS, starting from only 50 μg of protein per condition and analyzing only 40% of the obtained fractions. Using ChaFRADIC ...[more]

PMID: 23964590

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