TY - JOUR
T1 - Unsupervised Identification of Isotope-Labeled Peptides
AU - Goldford, Joshua E.
AU - Libourel, Igor G
N1 - Publisher Copyright:
© 2016 American Chemical Society.
PY - 2016/6/7
Y1 - 2016/6/7
N2 - In vivo isotopic labeling coupled with high-resolution proteomics is used to investigate primary metabolism in techniques such as stable isotope probing (protein-SIP) and peptide-based metabolic flux analysis (PMFA). Isotopic enrichment of carbon substrates and intracellular metabolism determine the distribution of isotopes within amino acids. The resulting amino acid mass distributions (AMDs) are convoluted into peptide mass distributions (PMDs) during protein synthesis. With no a priori knowledge on metabolic fluxes, the PMDs are therefore unknown. This complicates labeled peptide identification because prior knowledge on PMDs is used in all available peptide identification software. An automated framework for the identification and quantification of PMDs for nonuniformly labeled samples is therefore lacking. To unlock the potential of peptide labeling experiments for high-throughput flux analysis and other complex labeling experiments, an unsupervised peptide identification and quantification method was developed that uses discrete deconvolution of mass distributions of identified peptides to inform on the mass distributions of otherwise unidentifiable peptides. Uniformly 13C-labeled Escherichia coli protein was used to test the developed feature reconstruction and deconvolution algorithms. The peptide identification was validated by comparing MS2-identified peptides to peptides identified from PMDs using unlabeled E. coli protein. Nonuniformly labeled Glycine max protein was used to demonstrate the technology on a representative sample suitable for flux analysis. Overall, automatic peptide identification and quantification were comparable or superior to manual extraction, enabling proteomics-based technology for high-throughput flux analysis studies.
AB - In vivo isotopic labeling coupled with high-resolution proteomics is used to investigate primary metabolism in techniques such as stable isotope probing (protein-SIP) and peptide-based metabolic flux analysis (PMFA). Isotopic enrichment of carbon substrates and intracellular metabolism determine the distribution of isotopes within amino acids. The resulting amino acid mass distributions (AMDs) are convoluted into peptide mass distributions (PMDs) during protein synthesis. With no a priori knowledge on metabolic fluxes, the PMDs are therefore unknown. This complicates labeled peptide identification because prior knowledge on PMDs is used in all available peptide identification software. An automated framework for the identification and quantification of PMDs for nonuniformly labeled samples is therefore lacking. To unlock the potential of peptide labeling experiments for high-throughput flux analysis and other complex labeling experiments, an unsupervised peptide identification and quantification method was developed that uses discrete deconvolution of mass distributions of identified peptides to inform on the mass distributions of otherwise unidentifiable peptides. Uniformly 13C-labeled Escherichia coli protein was used to test the developed feature reconstruction and deconvolution algorithms. The peptide identification was validated by comparing MS2-identified peptides to peptides identified from PMDs using unlabeled E. coli protein. Nonuniformly labeled Glycine max protein was used to demonstrate the technology on a representative sample suitable for flux analysis. Overall, automatic peptide identification and quantification were comparable or superior to manual extraction, enabling proteomics-based technology for high-throughput flux analysis studies.
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U2 - 10.1021/acs.analchem.6b01703
DO - 10.1021/acs.analchem.6b01703
M3 - Article
C2 - 27145348
AN - SCOPUS:84974559959
SN - 0003-2700
VL - 88
SP - 6092
EP - 6099
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 11
ER -