Journal article
Neutron Encoded Labeling for Peptide Identification
Analytical chemistry (Washington), Vol.85(10), pp.5129-5137
05/21/2013
DOI: 10.1021/ac400476w
PMCID: PMC3827945
PMID: 23638792
Abstract
Metabolic labeling of cells using heavy amino acids is most commonly used for relative quantitation; however, partner mass shifts also detail the number of heavy amino acids contained within the precursor species. Here, we use a recently developed metabolic labeling technique, NeuCode (neutron encoding) stable isotope labeling with amino acids in cell culture (SILAC), which produces precursor partners spaced ~40 mDa apart to enable amino acid counting. We implement large scale counting of amino acids through a program, “Amino Acid Counter”, which determines the most likely combination of amino acids within a precursor based on NeuCode SILAC partner spacing and filters candidate peptide sequences during a database search using this information. Counting the number of lysine residues for precursors selected for MS/MS decreases the median number of candidate sequences from 44 to 14 as compared to an accurate mass search alone (20 ppm). Furthermore, the ability to co-isolate and fragment NeuCode SILAC partners enables counting of lysines in product ions, and when the information is used, the median number of candidates is reduced to 7. We then demonstrate counting leucine in addition to lysine results in a 6-fold decrease in search space, 43 to 7, when compared to an accurate mass search. We use this scheme to analyze a nanoLC-MS/MS experiment and demonstrate that accurate mass plus lysine and leucine counting reduces the number of candidate sequences to one for ~20% of all precursors selected, demonstrating an ability to identify precursors without MS/MS analysis.
Details
- Title: Subtitle
- Neutron Encoded Labeling for Peptide Identification
- Creators
- Christopher M Rose - Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United StatesAnna E Merrill - Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United StatesDerek J Bailey - Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United StatesAlexander S Hebert - Genome Center of Wisconsin, University of Wisconsin, Madison, Wisconsin 53706, United StatesMichael S Westphall - Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United StatesJoshua J Coon - Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, United States
- Resource Type
- Journal article
- Publication Details
- Analytical chemistry (Washington), Vol.85(10), pp.5129-5137
- DOI
- 10.1021/ac400476w
- PMID
- 23638792
- PMCID
- PMC3827945
- NLM abbreviation
- Anal Chem
- ISSN
- 0003-2700
- eISSN
- 1520-6882
- Grant note
- T32 GM008505 || GM / National Institute of General Medical Sciences : NIGMS R01 GM080148 || GM / National Institute of General Medical Sciences : NIGMS T32 HG002760 || HG / National Human Genome Research Institute : NHGRI
- Language
- English
- Date published
- 05/21/2013
- Academic Unit
- Pathology; Injury Prevention Research Center
- Record Identifier
- 9984047610302771
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