Protein identification in biomarker discovery studies based on
profiling of intact proteins: Why the challenge? Lesson gained
from breast cancer tissues

a 2012

Protein identification in biomarker discovery studies based on profiling of intact proteins: Why the challenge? Lesson gained from breast cancer tissues

BOUCHAL, Pavel; Monika DVOŘÁKOVÁ; Spiros D. GARBIS; Alexander SCHERL; Rudolf NENUTIL et al.

Základní údaje

Originální název

Protein identification in biomarker discovery studies based on profiling of intact proteins: Why the challenge? Lesson gained from breast cancer tissues

Autoři

BOUCHAL, Pavel; Monika DVOŘÁKOVÁ; Spiros D. GARBIS; Alexander SCHERL; Rudolf NENUTIL a Bořivoj VOJTĚŠEK

Vydání

60th Conference on Mass Spectrometry and Allied Topics, 2012

Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Obor

10600 1.6 Biological sciences

Stát vydavatele

Kanada

Utajení

není předmětem státního či obchodního tajemství

Odkazy

ASMS home page

Označené pro přenos do RIV

Organizační jednotka

Přírodovědecká fakulta

Klíčová slova anglicky

top-down proteomics; FT-MS; MALDI; SELDI; 3D separation; breast cancer

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 4. 4. 2013 18:10, doc. Mgr. Pavel Bouchal, Ph.D.

Anotace

V originále

We showed thate the use of simple combination of protein/peptide separation technique followed by MS/MS leads to high uncertainty in protein biomarker identification in biomarker discovery studies based on profiling of intact proteins. This is given namely by the fact that database molecular weight of proteins does not reflect their posttranslational modifications. Here we propose two protocols to overcome these bottlenecks: (i) For proteins larger than 6 kDa, we have developed a method combining IMAC column preseparation, reverse phase LC, MALDI/SELDI protein profiling, tricine SDS-PAGE, protein profile matching, trypsin digestion and MS/MS identification. (ii) For identification of proteins smaller than 6 kDa, we have optimized a "top-down" procedure where tricine SDS-PAGE is replaced by LC-MS/MS of proteins on LTQ Orbitrap Velos with the use "no enzyme" option during MS/MS ion search. This protocol enabled and identification of HNRNP A2/B1 protein variant correlating with estrogen receptor expression in breast cancer.

Návaznosti

GAP304/10/0868, projekt VaV

Název: Studium molekulárních mechanismů časného metastazování do lymfatických uzlin u karcinomu prsu nízkého stupně malignity pomocí proteomických technik

Investor: Grantová agentura ČR, Studium molekulárních mechanismů časného metastazování do lymfatických uzlin u karcinomu prsu nízkého stupně malignity pomocí proteomických technik

Citovat

BOUCHAL, Pavel; Monika DVOŘÁKOVÁ; Spiros D. GARBIS; Alexander SCHERL; Rudolf NENUTIL a Bořivoj VOJTĚŠEK. Protein identification in biomarker discovery studies based on profiling of intact proteins: Why the challenge? Lesson gained from breast cancer tissues. In 60th Conference on Mass Spectrometry and Allied Topics. 2012.

@proceedings{1094691,
   author = {Bouchal, Pavel and Dvořáková, Monika and Garbis, Spiros D. and Scherl, Alexander and Nenutil, Rudolf and Vojtěšek, Bořivoj},
   booktitle = {60th Conference on Mass Spectrometry and Allied Topics},
   keywords = {top-down proteomics; FT-MS; MALDI; SELDI; 3D separation; breast cancer},
   language = {eng},
   title = {Protein identification in biomarker discovery studies based on profiling of intact proteins: Why the challenge? Lesson gained from breast cancer tissues},
   url = {http://www.asms.org},
   year = {2012}
}

TY  - CONF
ID  - 1094691
AU  - Bouchal, Pavel - Dvořáková, Monika - Garbis, Spiros D. - Scherl, Alexander - Nenutil, Rudolf - Vojtěšek, Bořivoj
PY  - 2012
TI  - Protein identification in biomarker discovery studies based on profiling of intact proteins: Why the challenge? Lesson gained from breast cancer tissues
KW  - top-down proteomics
KW  - FT-MS
KW  - MALDI
KW  - SELDI
KW  - 3D separation
KW  - breast cancer
UR  - http://www.asms.org
N2  - We showed thate the use of simple combination of protein/peptide separation technique followed by MS/MS leads to high uncertainty in protein biomarker identification in biomarker discovery studies based on profiling of intact proteins. This is given namely by the fact that database molecular weight of proteins does not reflect their posttranslational modifications. Here we propose two protocols to overcome these bottlenecks: (i) For proteins larger than 6 kDa, we have developed a method combining IMAC column preseparation, reverse phase LC, MALDI/SELDI protein profiling, tricine SDS-PAGE, protein profile matching, trypsin digestion and MS/MS identification. (ii) For identification of proteins smaller than 6 kDa, we have optimized a "top-down" procedure where tricine SDS-PAGE is replaced by LC-MS/MS of proteins on LTQ Orbitrap Velos with the use "no enzyme" option during MS/MS ion search. This protocol enabled and identification of HNRNP A2/B1 protein variant correlating with estrogen receptor expression in breast cancer.
ER  -

BOUCHAL, Pavel; Monika DVOŘÁKOVÁ; Spiros D. GARBIS; Alexander SCHERL; Rudolf NENUTIL a Bořivoj VOJTĚŠEK. Protein identification in biomarker discovery studies based on profiling of intact proteins: Why the challenge? Lesson gained from breast cancer tissues. In \textit{60th Conference on Mass Spectrometry and Allied Topics}. 2012.

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