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Arginine methylation in yeast proteins during stationary-phase growth and heat shock

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Abstract

Arginine methyltransferases (RMTs) catalyze the methylation of arginine residues on proteins. We examined the effects of log-phase growth, stationary-phase growth, and heat shock on the formation of methylarginines on yeast proteins to determine if the conditions favor a particular type of methylation. Utilizing linear ion trap mass spectrometry, we identify methylarginines in wild-type and RMT deletion yeast strains using secondary product ion scans (MS3), and quantify the methylarginines using multiple reaction monitoring (MRM). Employing MS3 and isotopic incorporation, we demonstrate for the first time that Nη1, Nη2-dimethylarginine (sDMA) is present on yeast proteins, and make a detailed structural determination of the fragment ions from the spectra. Nη-monomethylarginine (ηMMA), Nδ-monomethylarginine (δMMA), Nη1, Nη1-dimethylarginine (aDMA), and sDMA were detected in RMT deletion yeast using MS3 and MRM with and without isotopic incorporation, suggesting that additional RMT enzymes remain to be discovered in yeast. The concentrations of ηMMA and δMMA decreased by half during heat shock and stationary phase compared to log-phase growth of wild-type yeast, whereas sDMA increased by as much as sevenfold and aDMA decreased by 11-fold. Therefore, upon entering stressful conditions like heat shock or stationary-phase growth, there is a net increase in sDMA and decreases in aDMA, ηMMA, and δMMA on yeast proteins.

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Abbreviations

CE:

Collision energy

CPS:

Counts per second

CXP:

Collision cell exit potential

DP:

Declustering potential

MRM:

Multiple reaction monitoring

MRM3 :

Multiple reaction monitoring cubed

MS2 :

Primary product ion spectrum

MS3 :

Secondary product ion spectrum

MS:

Mass spectrometry

UHPLC:

Ultra-high performance liquid chromatography

YEPD:

Yeast extract peptone dextrose

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Acknowledgments

The authors acknowledge support from the BC Proteomics Network Small Projects Health Research Grant (to A.F.), The University of British Columbia Doctoral Fellowships (to D.T. and M.I.V.), The Dr. Paul H.T. Thorlakson Foundation Fund and The Manitoba Medical Service Foundation Grant, University Research Grants Program (University of Manitoba), and the Natural Sciences and Engineering Research Council (NSERC) of Canada RGPIN-2015-06543 (to T.M.L). This manuscript is dedicated to the memory of Dr. Romuald Lakowski, who was a loving father, a mentor, and a dedicated scientist.

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Authors and Affiliations

  1. Pharmaceutical Analysis Laboratory, Faculty of Health Sciences, College of Pharmacy, The University of Manitoba, 750 McDermot Avenue, Winnipeg, Manitoba, R3E 0T5, Canada

    Ted M. Lakowski

  2. University of Massachusetts Medical School, Worcester, MA, USA

    Magnolia L. Pak

  3. Faculty of Pharmaceutical Sciences, The University of British Columbia, 2405 Wesbrook Mall, Vancouver, BC, V6T 1Z3, Canada

    András Szeitz, Dylan Thomas, Mynol I. Vhuiyan & Adam Frankel

  4. Pharmaceutical Institute, Gutenbergstr. 76, 24118, Kiel, Germany

    Bernd Clement

Authors
  1. Ted M. Lakowski
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  2. Magnolia L. Pak
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  3. András Szeitz
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  4. Dylan Thomas
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  5. Mynol I. Vhuiyan
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  6. Bernd Clement
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  7. Adam Frankel
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Correspondence to Ted M. Lakowski or Adam Frankel.

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Handling Editor: K. L. Bennett.

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Lakowski, T.M., Pak, M.L., Szeitz, A. et al. Arginine methylation in yeast proteins during stationary-phase growth and heat shock. Amino Acids 47, 2561–2571 (2015). https://doi.org/10.1007/s00726-015-2047-5

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