SP3 Protocol for Proteomic Plant Sample Preparation Prior
LC-MS/MS

J 2021

SP3 Protocol for Proteomic Plant Sample Preparation Prior LC-MS/MS

MIKULÁŠEK, Kamil, Hana KONEČNÁ, David POTĚŠIL, Renata HOLÁNKOVÁ, Jan HAVLIŠ et. al.

Basic information

Original name

SP3 Protocol for Proteomic Plant Sample Preparation Prior LC-MS/MS

Authors

MIKULÁŠEK, Kamil (203 Czech Republic, belonging to the institution), Hana KONEČNÁ (203 Czech Republic, belonging to the institution), David POTĚŠIL (203 Czech Republic, belonging to the institution), Renata HOLÁNKOVÁ (203 Czech Republic, belonging to the institution), Jan HAVLIŠ (203 Czech Republic, belonging to the institution) and Zbyněk ZDRÁHAL (203 Czech Republic, guarantor, belonging to the institution)

Edition

Frontiers in Plant Science, Frontiers Research Foundation, 2021, 1664-462X

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10611 Plant sciences, botany

Country of publisher

Switzerland

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

fulltext

Impact factor

Impact factor: 6.627

RIV identification code

RIV/00216224:14740/21:00121574

Organization unit

Central European Institute of Technology

DOI

http://dx.doi.org/10.3389/fpls.2021.635550

UT WoS

000631851900001

Keywords in English

bottom-up; protein cleanup; Arabidopsis thaliana; sodium dodecyl sulfate removal; single-pot solid-phase-enhanced sample preparation; carboxylated magnetic beads; filter-aided sample preparation; mass spectrometry

Abstract

V originále

Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the filter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal and protein digestion from Arabidopsis thaliana (AT) lysate. Finally, the optimized carboxylated SP3 workflow was benchmarked closely against the routine FASP. Ultimately, LC-MS/MS analyses revealed that regarding the number of identifications, number of missed cleavages, proteome coverage, repeatability, reduction of handling time, and cost per assay, the SP3 on carboxylated magnetic particles proved to be the best alternative for SDS and other contaminants removal from plant sample lysate. A robust and efficient 2-h SP3 protocol for a wide range of protein input is presented, benefiting from no need to adjust the amount of beads, binding and rinsing conditions, or digestion parameters.

Links

EF16_013/0001776, research and development project

Name: Česká infrastruktura pro integrativní strukturní biologii pro lidské zdraví

EF16_019/0000738, research and development project

Name: Centrum experimentální biologie rostlin

LM2018127, research and development project

Name: Česká infrastruktura pro integrativní strukturní biologii (Acronym: CIISB)

Investor: Ministry of Education, Youth and Sports of the CR

LM2018140, research and development project

Name: e-Infrastruktura CZ (Acronym: e-INFRA CZ)

Investor: Ministry of Education, Youth and Sports of the CR

Citovat

MIKULÁŠEK, Kamil, Hana KONEČNÁ, David POTĚŠIL, Renata HOLÁNKOVÁ, Jan HAVLIŠ and Zbyněk ZDRÁHAL. SP3 Protocol for Proteomic Plant Sample Preparation Prior LC-MS/MS. Frontiers in Plant Science. Frontiers Research Foundation, 2021, vol. 12, MAR, p. "635550", 12 pp. ISSN 1664-462X. Available from: https://dx.doi.org/10.3389/fpls.2021.635550.

@article{1768079,
   author = {Mikulášek, Kamil and Konečná, Hana and Potěšil, David and Holánková, Renata and Havliš, Jan and Zdráhal, Zbyněk},
   article_number = {MAR},
   doi = {http://dx.doi.org/10.3389/fpls.2021.635550},
   keywords = {bottom-up; protein cleanup; Arabidopsis thaliana; sodium dodecyl sulfate removal; single-pot solid-phase-enhanced sample preparation; carboxylated magnetic beads; filter-aided sample preparation; mass spectrometry},
   language = {eng},
   issn = {1664-462X},
   journal = {Frontiers in Plant Science},
   title = {SP3 Protocol for Proteomic Plant Sample Preparation Prior LC-MS/MS},
   url = {https://www.frontiersin.org/articles/10.3389/fpls.2021.635550/full},
   volume = {12},
   year = {2021}
}

TY  - JOUR
ID  - 1768079
AU  - Mikulášek, Kamil - Konečná, Hana - Potěšil, David - Holánková, Renata - Havliš, Jan - Zdráhal, Zbyněk
PY  - 2021
TI  - SP3 Protocol for Proteomic Plant Sample Preparation Prior LC-MS/MS
JF  - Frontiers in Plant Science
VL  - 12
IS  - MAR
SP  - "635550"
EP  - "635550"
PB  - Frontiers Research Foundation
SN  - 1664462X
KW  - bottom-up
KW  - protein cleanup
KW  - Arabidopsis thaliana
KW  - sodium dodecyl sulfate removal
KW  - single-pot solid-phase-enhanced sample preparation
KW  - carboxylated magnetic beads
KW  - filter-aided sample preparation
KW  - mass spectrometry
UR  - https://www.frontiersin.org/articles/10.3389/fpls.2021.635550/full
N2  - Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the filter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal and protein digestion from Arabidopsis thaliana (AT) lysate. Finally, the optimized carboxylated SP3 workflow was benchmarked closely against the routine FASP. Ultimately, LC-MS/MS analyses revealed that regarding the number of identifications, number of missed cleavages, proteome coverage, repeatability, reduction of handling time, and cost per assay, the SP3 on carboxylated magnetic particles proved to be the best alternative for SDS and other contaminants removal from plant sample lysate. A robust and efficient 2-h SP3 protocol for a wide range of protein input is presented, benefiting from no need to adjust the amount of beads, binding and rinsing conditions, or digestion parameters.
ER  -

MIKULÁŠEK, Kamil, Hana KONEČNÁ, David POTĚŠIL, Renata HOLÁNKOVÁ, Jan HAVLIŠ and Zbyněk ZDRÁHAL. SP3 Protocol for Proteomic Plant Sample Preparation Prior LC-MS/MS. \textit{Frontiers in Plant Science}. Frontiers Research Foundation, 2021, vol.~12, MAR, p.~''635550'', 12 pp. ISSN~1664-462X. Available from: https://dx.doi.org/10.3389/fpls.2021.635550.

Detailed Information on Publication Record