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Improved single-step enrichment methods of cross-linked products for protein structure analysis and protein interaction mapping

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Abstract

Chemical cross-linking/mass spectrometry (MS) is gradually developing into a routine method to investigate protein conformation and to decipher protein interaction networks. To increase identification rates of the frequently low abundant cross-linked products in LC/MS/MS experiments, fast and reliable sample preparation protocols are indispensable. We present simplified solid phase extraction methods using C18/SCX StageTips and mixed-mode OASIS MCX cartridges for a single-step enrichment of cross-linked products prior to LC/MS/MS analysis. Our improved protocols result in 3.5 to 4.6 times higher numbers of cross-link identifications for the model protein bovine serum albumin compared to non-processed samples.

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Abbreviations

BS3 :

Bis(sulfosuccinimidyl)suberate

BSA:

Bovine serum albumin

CID:

Collision-induced dissociation

ESI:

Electrospray ionization

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HPLC:

High-performance liquid chromatography

LC:

Liquid chromatography

LTQ:

Linear ion trap (Thermo Fisher Scientific)

MCX:

Mixed mode cation exchange (Waters)

MeOH:

Methanol

MS:

Mass spectrometry

MS/MS:

Tandem mass spectrometry

RP:

Reversed phase

SCX:

Strong cation exchange chromatography

SEC:

Size exclusion chromatography

SPE:

Solid phase extraction

TFA:

Trifluoroacetic acid

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Acknowledgements

AS is supported by the DFG (project Si 867/15-2) and the region of Saxony Anhalt. The authors are indebted to Mr. Thomas Piotrowski for constructing the SPE apparatus (ESM, Fig. S12).

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Authors and Affiliations

  1. Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-St. 4, 06120, Halle (Saale), Germany

    Rico Schmidt & Andrea Sinz

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  1. Rico Schmidt
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  2. Andrea Sinz
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Correspondence to Andrea Sinz.

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Schmidt, R., Sinz, A. Improved single-step enrichment methods of cross-linked products for protein structure analysis and protein interaction mapping. Anal Bioanal Chem 409, 2393–2400 (2017). https://doi.org/10.1007/s00216-017-0185-1

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  • DOI: https://doi.org/10.1007/s00216-017-0185-1

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