Quantitative proteomics

Measuring protein synthesis using 15N amino acid labeling in pancreatic cancer cells

Yingchun Zhao, Wai Nang Paul Lee, Shu Lim, Vay Liang Go, Jing Xiao, Rui Cao, Hengwei Zhang, Robert R. Recker, Gary Guishan Xiao

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Pancreatic cancer MIA PaCa cells were cultured in the presence and absence of 15N amino acids mixture for 72 h. During protein synthesis, the incorporation of 15N amino acids results in a new mass isotopomer distribution in protein, which is approximated by the concatenation of two binomial distributions of 13C and 15N. The fraction of protein synthesis (FSR) can thus be determined from the relative intensities of the"labeled" (new) and the "unlabeled" (old) spectra. Six prominent spots were picked from 2-D gels of proteins from lysates of cells cultured in 0% (control), 50%, and 33% 15N enriched media. These protein spots were digested and analyzed with matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. The isotopomer distribution of peptides after labeling can be fully accounted for by the labeled (new) and unlabeled (old) peptides. The ratio of the new and old peptide fractions was determined using multiple regression analysis of the observed spectrum as a linear combination of the expected new and the old spectra. The fractional protein synthesis rates calculated from such ratios of the same peptide from cells grown in 50% and 33% 15N amino acid enrichments were comparable to each other. The FSR of these six identified proteins ranged between 44 and 76%.

Original languageEnglish
Pages (from-to)764-771
Number of pages8
JournalAnalytical Chemistry
Volume81
Issue number2
DOIs
StatePublished - Jan 15 2009

Fingerprint

Labeling
Cells
Amino Acids
Proteins
Peptides
Proteomics
Regression analysis
Ionization
Mass spectrometry
Desorption
Gels
Lasers

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

Cite this

Zhao, Y., Lee, W. N. P., Lim, S., Go, V. L., Xiao, J., Cao, R., ... Xiao, G. G. (2009). Quantitative proteomics: Measuring protein synthesis using 15N amino acid labeling in pancreatic cancer cells. Analytical Chemistry, 81(2), 764-771. https://doi.org/10.1021/ac801905g

Quantitative proteomics : Measuring protein synthesis using 15N amino acid labeling in pancreatic cancer cells. / Zhao, Yingchun; Lee, Wai Nang Paul; Lim, Shu; Go, Vay Liang; Xiao, Jing; Cao, Rui; Zhang, Hengwei; Recker, Robert R.; Xiao, Gary Guishan.

In: Analytical Chemistry, Vol. 81, No. 2, 15.01.2009, p. 764-771.

Research output: Contribution to journalArticle

Zhao, Yingchun ; Lee, Wai Nang Paul ; Lim, Shu ; Go, Vay Liang ; Xiao, Jing ; Cao, Rui ; Zhang, Hengwei ; Recker, Robert R. ; Xiao, Gary Guishan. / Quantitative proteomics : Measuring protein synthesis using 15N amino acid labeling in pancreatic cancer cells. In: Analytical Chemistry. 2009 ; Vol. 81, No. 2. pp. 764-771.
@article{9b8027809826489cb836413de9729f47,
title = "Quantitative proteomics: Measuring protein synthesis using 15N amino acid labeling in pancreatic cancer cells",
abstract = "Pancreatic cancer MIA PaCa cells were cultured in the presence and absence of 15N amino acids mixture for 72 h. During protein synthesis, the incorporation of 15N amino acids results in a new mass isotopomer distribution in protein, which is approximated by the concatenation of two binomial distributions of 13C and 15N. The fraction of protein synthesis (FSR) can thus be determined from the relative intensities of the{"}labeled{"} (new) and the {"}unlabeled{"} (old) spectra. Six prominent spots were picked from 2-D gels of proteins from lysates of cells cultured in 0{\%} (control), 50{\%}, and 33{\%} 15N enriched media. These protein spots were digested and analyzed with matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. The isotopomer distribution of peptides after labeling can be fully accounted for by the labeled (new) and unlabeled (old) peptides. The ratio of the new and old peptide fractions was determined using multiple regression analysis of the observed spectrum as a linear combination of the expected new and the old spectra. The fractional protein synthesis rates calculated from such ratios of the same peptide from cells grown in 50{\%} and 33{\%} 15N amino acid enrichments were comparable to each other. The FSR of these six identified proteins ranged between 44 and 76{\%}.",
author = "Yingchun Zhao and Lee, {Wai Nang Paul} and Shu Lim and Go, {Vay Liang} and Jing Xiao and Rui Cao and Hengwei Zhang and Recker, {Robert R.} and Xiao, {Gary Guishan}",
year = "2009",
month = "1",
day = "15",
doi = "10.1021/ac801905g",
language = "English",
volume = "81",
pages = "764--771",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "2",

}

TY - JOUR

T1 - Quantitative proteomics

T2 - Measuring protein synthesis using 15N amino acid labeling in pancreatic cancer cells

AU - Zhao, Yingchun

AU - Lee, Wai Nang Paul

AU - Lim, Shu

AU - Go, Vay Liang

AU - Xiao, Jing

AU - Cao, Rui

AU - Zhang, Hengwei

AU - Recker, Robert R.

AU - Xiao, Gary Guishan

PY - 2009/1/15

Y1 - 2009/1/15

N2 - Pancreatic cancer MIA PaCa cells were cultured in the presence and absence of 15N amino acids mixture for 72 h. During protein synthesis, the incorporation of 15N amino acids results in a new mass isotopomer distribution in protein, which is approximated by the concatenation of two binomial distributions of 13C and 15N. The fraction of protein synthesis (FSR) can thus be determined from the relative intensities of the"labeled" (new) and the "unlabeled" (old) spectra. Six prominent spots were picked from 2-D gels of proteins from lysates of cells cultured in 0% (control), 50%, and 33% 15N enriched media. These protein spots were digested and analyzed with matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. The isotopomer distribution of peptides after labeling can be fully accounted for by the labeled (new) and unlabeled (old) peptides. The ratio of the new and old peptide fractions was determined using multiple regression analysis of the observed spectrum as a linear combination of the expected new and the old spectra. The fractional protein synthesis rates calculated from such ratios of the same peptide from cells grown in 50% and 33% 15N amino acid enrichments were comparable to each other. The FSR of these six identified proteins ranged between 44 and 76%.

AB - Pancreatic cancer MIA PaCa cells were cultured in the presence and absence of 15N amino acids mixture for 72 h. During protein synthesis, the incorporation of 15N amino acids results in a new mass isotopomer distribution in protein, which is approximated by the concatenation of two binomial distributions of 13C and 15N. The fraction of protein synthesis (FSR) can thus be determined from the relative intensities of the"labeled" (new) and the "unlabeled" (old) spectra. Six prominent spots were picked from 2-D gels of proteins from lysates of cells cultured in 0% (control), 50%, and 33% 15N enriched media. These protein spots were digested and analyzed with matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. The isotopomer distribution of peptides after labeling can be fully accounted for by the labeled (new) and unlabeled (old) peptides. The ratio of the new and old peptide fractions was determined using multiple regression analysis of the observed spectrum as a linear combination of the expected new and the old spectra. The fractional protein synthesis rates calculated from such ratios of the same peptide from cells grown in 50% and 33% 15N amino acid enrichments were comparable to each other. The FSR of these six identified proteins ranged between 44 and 76%.

UR - http://www.scopus.com/inward/record.url?scp=60549095613&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=60549095613&partnerID=8YFLogxK

U2 - 10.1021/ac801905g

DO - 10.1021/ac801905g

M3 - Article

VL - 81

SP - 764

EP - 771

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 2

ER -