Abstract
Mass spectrometry has become indispensable for peptide and protein quantification in proteomics studies. When proteomics technologies are applied to understand the biology of plants, two-dimensional gel electrophoresis is still the prevalent method for protein fractionation, identification, and quantitation. In the present work, we have used LC-MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of Ricinus communis seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in-solution, and the same amount of peptides was labeled with ICPL and iTRAQ tags in two orders (forward and reverse). Each sample was submitted to nanoLC coupled to an LTQ-Orbitrap high-resolution mass spectrometer. Comparing labeling performance, iTRAQ was able to label 99.8% of all identified unique peptides, while 94.1% were labeled by ICPL. After statistical analysis, it was possible to quantify 309 (ICPL) and 321 (iTRAQ) proteins, from which 95 are specific to ICPL, 107 to iTRAQ, and 214 common to both labeling strategies. We noted that the iTRAQ quantification could be influenced by the tag. Even though the efficiency of the iTRAQ and ICPL in protein quantification depends on several parameters, both labeling methods were able to successfully quantify proteins present in the endosperm of castor bean during seed development and, when combined, increase the number of quantified proteins.
| Original language | English |
|---|---|
| Pages (from-to) | 3046-3052 |
| Number of pages | 7 |
| Journal | Journal of Proteome Research |
| Volume | 11 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - May 4 2012 |
Fingerprint
Keywords
- high-resolution mass spectrometry
- ICPL
- iTRAQ
- plant proteomics
- quantitative proteomics
ASJC Scopus subject areas
- Biochemistry
- Chemistry(all)
Cite this
Performance of isobaric and isotopic labeling in quantitative plant proteomics. / Nogueira, Fábio C S; Palmisano, Giuseppe; Schwämmle, Veit; Campos, Francisco A P; Larsen, Martin R.; Domont, Gilberto B.; Roepstorff, Peter.
In: Journal of Proteome Research, Vol. 11, No. 5, 04.05.2012, p. 3046-3052.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Performance of isobaric and isotopic labeling in quantitative plant proteomics
AU - Nogueira, Fábio C S
AU - Palmisano, Giuseppe
AU - Schwämmle, Veit
AU - Campos, Francisco A P
AU - Larsen, Martin R.
AU - Domont, Gilberto B.
AU - Roepstorff, Peter
PY - 2012/5/4
Y1 - 2012/5/4
N2 - Mass spectrometry has become indispensable for peptide and protein quantification in proteomics studies. When proteomics technologies are applied to understand the biology of plants, two-dimensional gel electrophoresis is still the prevalent method for protein fractionation, identification, and quantitation. In the present work, we have used LC-MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of Ricinus communis seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in-solution, and the same amount of peptides was labeled with ICPL and iTRAQ tags in two orders (forward and reverse). Each sample was submitted to nanoLC coupled to an LTQ-Orbitrap high-resolution mass spectrometer. Comparing labeling performance, iTRAQ was able to label 99.8% of all identified unique peptides, while 94.1% were labeled by ICPL. After statistical analysis, it was possible to quantify 309 (ICPL) and 321 (iTRAQ) proteins, from which 95 are specific to ICPL, 107 to iTRAQ, and 214 common to both labeling strategies. We noted that the iTRAQ quantification could be influenced by the tag. Even though the efficiency of the iTRAQ and ICPL in protein quantification depends on several parameters, both labeling methods were able to successfully quantify proteins present in the endosperm of castor bean during seed development and, when combined, increase the number of quantified proteins.
AB - Mass spectrometry has become indispensable for peptide and protein quantification in proteomics studies. When proteomics technologies are applied to understand the biology of plants, two-dimensional gel electrophoresis is still the prevalent method for protein fractionation, identification, and quantitation. In the present work, we have used LC-MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of Ricinus communis seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in-solution, and the same amount of peptides was labeled with ICPL and iTRAQ tags in two orders (forward and reverse). Each sample was submitted to nanoLC coupled to an LTQ-Orbitrap high-resolution mass spectrometer. Comparing labeling performance, iTRAQ was able to label 99.8% of all identified unique peptides, while 94.1% were labeled by ICPL. After statistical analysis, it was possible to quantify 309 (ICPL) and 321 (iTRAQ) proteins, from which 95 are specific to ICPL, 107 to iTRAQ, and 214 common to both labeling strategies. We noted that the iTRAQ quantification could be influenced by the tag. Even though the efficiency of the iTRAQ and ICPL in protein quantification depends on several parameters, both labeling methods were able to successfully quantify proteins present in the endosperm of castor bean during seed development and, when combined, increase the number of quantified proteins.
KW - high-resolution mass spectrometry
KW - ICPL
KW - iTRAQ
KW - plant proteomics
KW - quantitative proteomics
UR - http://www.scopus.com/inward/record.url?scp=84860641713&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84860641713&partnerID=8YFLogxK
U2 - 10.1021/pr300192f
DO - 10.1021/pr300192f
M3 - Article
C2 - 22452248
AN - SCOPUS:84860641713
VL - 11
SP - 3046
EP - 3052
JO - Journal of Proteome Research
JF - Journal of Proteome Research
SN - 1535-3893
IS - 5
ER -