Publication

Ariadne's Thread in the Analytical Labyrinth of Membrane Proteins: Integration of Targeted and Shotgun Proteomics for Global Absolute Quantification of Membrane Proteins

Antelo-Varela, M., Bartel, J., Quesada-Ganuza, A., Appel, K., Bernal-Cabas, M., Sura, T., Otto, A., Rasmussen, M., van Dijl, J. M., Nielsen, A., Maass, S. & Becher, D., 19-Sep-2019, In : Analytical Chemistry. 91, 18, p. 11972-11980 9 p.

Research output: Contribution to journalArticleAcademicpeer-review

APA

Antelo-Varela, M., Bartel, J., Quesada-Ganuza, A., Appel, K., Bernal-Cabas, M., Sura, T., ... Becher, D. (2019). Ariadne's Thread in the Analytical Labyrinth of Membrane Proteins: Integration of Targeted and Shotgun Proteomics for Global Absolute Quantification of Membrane Proteins. Analytical Chemistry, 91(18), 11972-11980. https://doi.org/10.1021/acs.analchem.9b02869

Author

Antelo-Varela, Minia ; Bartel, Juergen ; Quesada-Ganuza, Ane ; Appel, Karen ; Bernal-Cabas, Margarita ; Sura, Thomas ; Otto, Andreas ; Rasmussen, Michael ; van Dijl, Jan Maarten ; Nielsen, Allan ; Maass, Sandra ; Becher, Doerte. / Ariadne's Thread in the Analytical Labyrinth of Membrane Proteins : Integration of Targeted and Shotgun Proteomics for Global Absolute Quantification of Membrane Proteins. In: Analytical Chemistry. 2019 ; Vol. 91, No. 18. pp. 11972-11980.

Harvard

Antelo-Varela, M, Bartel, J, Quesada-Ganuza, A, Appel, K, Bernal-Cabas, M, Sura, T, Otto, A, Rasmussen, M, van Dijl, JM, Nielsen, A, Maass, S & Becher, D 2019, 'Ariadne's Thread in the Analytical Labyrinth of Membrane Proteins: Integration of Targeted and Shotgun Proteomics for Global Absolute Quantification of Membrane Proteins', Analytical Chemistry, vol. 91, no. 18, pp. 11972-11980. https://doi.org/10.1021/acs.analchem.9b02869

Standard

Ariadne's Thread in the Analytical Labyrinth of Membrane Proteins : Integration of Targeted and Shotgun Proteomics for Global Absolute Quantification of Membrane Proteins. / Antelo-Varela, Minia; Bartel, Juergen; Quesada-Ganuza, Ane; Appel, Karen; Bernal-Cabas, Margarita; Sura, Thomas; Otto, Andreas; Rasmussen, Michael; van Dijl, Jan Maarten; Nielsen, Allan; Maass, Sandra; Becher, Doerte.

In: Analytical Chemistry, Vol. 91, No. 18, 19.09.2019, p. 11972-11980.

Research output: Contribution to journalArticleAcademicpeer-review

Vancouver

Antelo-Varela M, Bartel J, Quesada-Ganuza A, Appel K, Bernal-Cabas M, Sura T et al. Ariadne's Thread in the Analytical Labyrinth of Membrane Proteins: Integration of Targeted and Shotgun Proteomics for Global Absolute Quantification of Membrane Proteins. Analytical Chemistry. 2019 Sep 19;91(18):11972-11980. https://doi.org/10.1021/acs.analchem.9b02869


BibTeX

@article{c31d4d915b7c404fb2766a933c7ce500,
title = "Ariadne's Thread in the Analytical Labyrinth of Membrane Proteins: Integration of Targeted and Shotgun Proteomics for Global Absolute Quantification of Membrane Proteins",
abstract = "The field of systems biology has been rapidly developing in the past decade. However, the data produced by {"}omits{"} approaches is lagging behind the requirements of this field, especially when it comes to absolute abundances of membrane proteins. In the present study, a novel approach for large-scale absolute quantification of this challenging subset of proteins has been established and evaluated using osmotic stress management in the Gram-positive model bacterium Bacillus subtilis as proof-of-principle precedent. Selected membrane proteins were labeled using a SNAP-tag, which allowed us to visually inspect the enrichment of the membrane fraction by immunoassays. Absolute membrane protein concentrations were determined via shotgun proteomics by spiking crude membrane extracts of chromosomally SNAP-tagged and wild-type B. subtilis strains with protein standards of known concentration. Shotgun data was subsequently calibrated by targeted mass Snotgun-his Targeted MY spectrometry using SNAP as an anchor protein, and an enrichment factor was calculated in order to obtain membrane protein copy numbers per square micrometer. The presented approach enabled the accurate determination of physiological changes resulting from imposed hyperosmotic stress, thereby offering a clear visualization of alterations in membrane protein arrangements and shedding light on putative membrane complexes. This straightforward and cost-effective methodology for quantitative proteome studies can be implemented by any research group with mass spectrometry expertise. Importantly, it can be applied to the full spectrum of physiologically relevant conditions, ranging from environmental stresses to the biotechnological production of small molecules and proteins, a field heavily relying on B. subtilis secretion capabilities.",
keywords = "SAMPLE PREPARATION METHOD, BACILLUS-SUBTILIS, MOLECULES, CELL",
author = "Minia Antelo-Varela and Juergen Bartel and Ane Quesada-Ganuza and Karen Appel and Margarita Bernal-Cabas and Thomas Sura and Andreas Otto and Michael Rasmussen and {van Dijl}, {Jan Maarten} and Allan Nielsen and Sandra Maass and Doerte Becher",
year = "2019",
month = "9",
day = "19",
doi = "10.1021/acs.analchem.9b02869",
language = "English",
volume = "91",
pages = "11972--11980",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "AMER CHEMICAL SOC INC",
number = "18",

}

RIS

TY - JOUR

T1 - Ariadne's Thread in the Analytical Labyrinth of Membrane Proteins

T2 - Integration of Targeted and Shotgun Proteomics for Global Absolute Quantification of Membrane Proteins

AU - Antelo-Varela, Minia

AU - Bartel, Juergen

AU - Quesada-Ganuza, Ane

AU - Appel, Karen

AU - Bernal-Cabas, Margarita

AU - Sura, Thomas

AU - Otto, Andreas

AU - Rasmussen, Michael

AU - van Dijl, Jan Maarten

AU - Nielsen, Allan

AU - Maass, Sandra

AU - Becher, Doerte

PY - 2019/9/19

Y1 - 2019/9/19

N2 - The field of systems biology has been rapidly developing in the past decade. However, the data produced by "omits" approaches is lagging behind the requirements of this field, especially when it comes to absolute abundances of membrane proteins. In the present study, a novel approach for large-scale absolute quantification of this challenging subset of proteins has been established and evaluated using osmotic stress management in the Gram-positive model bacterium Bacillus subtilis as proof-of-principle precedent. Selected membrane proteins were labeled using a SNAP-tag, which allowed us to visually inspect the enrichment of the membrane fraction by immunoassays. Absolute membrane protein concentrations were determined via shotgun proteomics by spiking crude membrane extracts of chromosomally SNAP-tagged and wild-type B. subtilis strains with protein standards of known concentration. Shotgun data was subsequently calibrated by targeted mass Snotgun-his Targeted MY spectrometry using SNAP as an anchor protein, and an enrichment factor was calculated in order to obtain membrane protein copy numbers per square micrometer. The presented approach enabled the accurate determination of physiological changes resulting from imposed hyperosmotic stress, thereby offering a clear visualization of alterations in membrane protein arrangements and shedding light on putative membrane complexes. This straightforward and cost-effective methodology for quantitative proteome studies can be implemented by any research group with mass spectrometry expertise. Importantly, it can be applied to the full spectrum of physiologically relevant conditions, ranging from environmental stresses to the biotechnological production of small molecules and proteins, a field heavily relying on B. subtilis secretion capabilities.

AB - The field of systems biology has been rapidly developing in the past decade. However, the data produced by "omits" approaches is lagging behind the requirements of this field, especially when it comes to absolute abundances of membrane proteins. In the present study, a novel approach for large-scale absolute quantification of this challenging subset of proteins has been established and evaluated using osmotic stress management in the Gram-positive model bacterium Bacillus subtilis as proof-of-principle precedent. Selected membrane proteins were labeled using a SNAP-tag, which allowed us to visually inspect the enrichment of the membrane fraction by immunoassays. Absolute membrane protein concentrations were determined via shotgun proteomics by spiking crude membrane extracts of chromosomally SNAP-tagged and wild-type B. subtilis strains with protein standards of known concentration. Shotgun data was subsequently calibrated by targeted mass Snotgun-his Targeted MY spectrometry using SNAP as an anchor protein, and an enrichment factor was calculated in order to obtain membrane protein copy numbers per square micrometer. The presented approach enabled the accurate determination of physiological changes resulting from imposed hyperosmotic stress, thereby offering a clear visualization of alterations in membrane protein arrangements and shedding light on putative membrane complexes. This straightforward and cost-effective methodology for quantitative proteome studies can be implemented by any research group with mass spectrometry expertise. Importantly, it can be applied to the full spectrum of physiologically relevant conditions, ranging from environmental stresses to the biotechnological production of small molecules and proteins, a field heavily relying on B. subtilis secretion capabilities.

KW - SAMPLE PREPARATION METHOD

KW - BACILLUS-SUBTILIS

KW - MOLECULES

KW - CELL

U2 - 10.1021/acs.analchem.9b02869

DO - 10.1021/acs.analchem.9b02869

M3 - Article

VL - 91

SP - 11972

EP - 11980

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 18

ER -

ID: 98525501