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Multidimensional nano-HPLC coupled with tandem mass spectrometry for analyzing biotinylated proteins

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Abstract

Multidimensional high-performance liquid chromatography (HPLC) is a key method in shotgun proteomics approaches for analyzing highly complex protein mixtures by complementary chromatographic separation principles. Here, we describe an integrated 3D-nano-HPLC/nano-electrospray ionization quadrupole time-of-flight mass spectrometry system that allows an enzymatic digestion of proteins followed by an enrichment and subsequent separation of the created peptide mixtures. The online 3D-nano-HPLC system is composed of a monolithic trypsin reactor in the first dimension, a monolithic affinity column with immobilized monomeric avidin in the second dimension, and a reversed phase C18 HPLC-Chip in the third dimension that is coupled to a nano-ESI-Q-TOF mass spectrometer. The 3D-LC/MS setup is exemplified for the identification of biotinylated proteins from a simple protein mixture. Additionally, we describe an online 2D-nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS setup for the enrichment, separation, and identification of cross-linked, biotinylated species from chemical cross-linking of cytochrome c and a calmodulin/peptide complex using a novel trifunctional cross-linker with two amine-reactive groups and a biotin label.

Schematic representations of the online 3D-nano-HPLC/nano-ESI-Q-TOF-MS/MS setup; LP loading pump, NP nano-pump

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Abbreviations

ACN:

Acetonitrile

BSA:

Bovine serum albumin

CaM:

Calmodulin

DMSO:

Dimethyl sulfoxide

ESI-Q-TOF:

Electrospray ionization quadrupole time-of-flight

FA:

Formic acid

HEPES:

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

IMER:

Immobilized monolithic enzyme reactor

MACMA:

Monolithic affinity column with immobilized monomeric avidin

MS:

Mass spectrometry

MS/MS:

Tandem mass spectrometry

NHS:

N-hydroxysuccinimide

RP:

Reversed phase

skMLCK:

Skeletal muscle myosin light chain kinase

TFA:

Trifluoroacetic acid

TOF:

Time-of-flight

Tris–HCl:

Tris(hydroxymethyl) aminomethane hydrochloride

Ugi-4CR:

Ugi-4-component reaction

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Acknowledgments

J.S. acknowledges the “Mass Spec Research Summer 2011” award (Agilent Technologies) of the DGMS (German Society for Mass Spectrometry). The authors thank Dr. C.H. Ihling for assistance with MS and Dr. O. Jahn for synthesis of the skMLCK peptide. J.S. thanks Dr. A. Svatoš and Dr. A. Muck for the introduction to monolithic supports.

Author information

Authors and Affiliations

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

    Jens Sproß & Andrea Sinz

  2. Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120, Halle (Saale), Germany

    Sebastian Brauch & Bernhard Westermann

  3. Agilent Technologies, Hewlett-Packard-Str. 8, 76337, Waldbronn, Germany

    Friedrich Mandel, Moritz Wagner & Stephan Buckenmaier

Authors
  1. Jens Sproß
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  2. Sebastian Brauch
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  3. Friedrich Mandel
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  4. Moritz Wagner
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  5. Stephan Buckenmaier
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  6. Bernhard Westermann
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  7. Andrea Sinz
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Corresponding authors

Correspondence to Jens Sproß or Andrea Sinz.

Additional information

Published in the topical collection Monolithic Columns in Liquid Phase Separations with guest editor Luis A. Colon.

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Sproß, J., Brauch, S., Mandel, F. et al. Multidimensional nano-HPLC coupled with tandem mass spectrometry for analyzing biotinylated proteins. Anal Bioanal Chem 405, 2163–2173 (2013). https://doi.org/10.1007/s00216-012-6057-9

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  • DOI: https://doi.org/10.1007/s00216-012-6057-9

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