A lysis-derived PMSF and orthovanadate oxidant defines a distinct cysteine-engagement profile

  • Cell Chem Biol. 2026 Aug 31:S2451-9456(26)00292-8. doi: 10.1016/j.chembiol.2026.08.005.
Bethany E Schaffer  1 ,  Jung Seung Nam  2 ,  Ceci A Cohen  3 ,  Clementina Mesaros  4 ,  Robert W Myers  5 ,  Leigh Baxt  5 ,  Kathryn E Wellen  6 ,  Lewis C Cantley  7 ,  Noah Dephoure  8 ,  Nathaniel W Snyder  9 ,  Iok In Christine Chio  10 ,  John Blenis  11
Affiliations
  • 1. Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA; Department of Pharmacology, Weill Cornell Medicine, New York, NY, USA. Electronic address: [email protected].
  • 2. Institute for Cancer Genetics, Department of Genetics & Development, Columbia University Irving Medical Center, New York, NY, USA; Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA.
  • 3. Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA; Department of Pharmacology, Weill Cornell Medicine, New York, NY, USA.
  • 4. Department of Systems Pharmacology and Translational Therapeutics (SPATT), University of Pennsylvania, Philadelphia, PA, USA.
  • 5. Sanders Tri-Institutional Therapeutics Discovery Institute, New York, NY, USA.
  • 6. Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, USA; Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA.
  • 7. Department of Cell Biology, Harvard Medical School, Boston, MA, USA; Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
  • 8. Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA; Department of Biochemistry, Weill Cornell Medicine, Cornell University, New York, NY, USA.
  • 9. Department of Cardiovascular Sciences, Aging + Cardiovascular Discovery Center, Lewis Katz School of Medicine, Temple University, Philadelphia, PA, USA.
  • 10. Institute for Cancer Genetics, Department of Genetics & Development, Columbia University Irving Medical Center, New York, NY, USA. Electronic address: [email protected].
  • 11. Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA; Department of Pharmacology, Weill Cornell Medicine, New York, NY, USA; Department of Biochemistry, Weill Cornell Medicine, Cornell University, New York, NY, USA. Electronic address: [email protected].
PMID: 42673962 DOI: 10.1016/j.chembiol.2026.08.005
Abstract

Reactive cysteines serve important functions in proteins, and characterizing their engagement by different electrophiles facilitates biological discovery and covalent drug development. Here, we show that the common lysis buffer components phenylmethylsulfonyl fluoride (PMSF) and orthovanadate generate a lysis-derived oxidant that engages cysteines during Cell Lysis. This oxidant sulfonylates N-acetyl-D-glucosamine kinase (NAGK) C217, producing a mobility shift on SDS-PAGE. C217 lies within the ATP-binding pocket, and a C217S mutant exhibits reduced ATP affinity and enzymatic activity. Competitive iodoacetamide-alkyne activity-based protein profiling (IAA-ABPP) chemoproteomics further showed that the PMSF/orthovanadate oxidant defines a cysteine-engagement profile that partially differs from that of pervanadate. These findings reveal an unrecognized source of chemical reactivity during protein extraction that expands the toolkit for cysteine-engagement profiling and underscores how sample preparation chemistry shapes chemoproteomic measurements.

Keywords
N-acetyl-D-glucosamine kinase; NAGK; PMSF; cysteine oxidation; cysteine reactivity; orthovanadate; phenylmethylsulfonyl fluoride.
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