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

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