Investigating Phosphorylation Patterns of the Ion Channel TRPM7 Using Multiple Extraction and Enrichment Techniques Reveals New Phosphosites

• Published in: Journal of the American Society for Mass Spectrometry Vol. 30; no. 8; pp. 1359 - 1367
• Main Authors: Nguyen, Thu T. A., Li, Wenping, Park, Thomas J., Gong, Liang-Wei, Cologna, Stephanie M.
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.08.2019; Springer Nature B.V
• Subjects: Amino Acid Sequence; Analytical Chemistry; Bioinformatics; Biotechnology; Chemistry; Chemistry and Materials Science; Chromatography, Liquid; Focus: Emerging Investigators: Research Article; HEK293 Cells; Humans; Ion channels; Kinases; Mass spectrometry; Organic Chemistry; Phosphopeptides - analysis; Phosphorylation; Protein Serine-Threonine Kinases - chemistry; Proteins; Proteomics; Solubilization; Tandem Mass Spectrometry; Titanium dioxide; TRPM Cation Channels - chemistry; Phosphorylation; S-Trap; Post-translational modifications; Phosphoproteomics; SCAD; TRPM7; Peptide enrichment; Membrane proteins
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1007/s13361-019-02223-5

The study of membrane proteins, and in particular ion channels, is crucial to understanding cellular function. Mass spectrometry-based approaches including bottom-up strategies to study membrane proteins have been successful yet still can remain challenging. In this study, we sought to evaluate the phosphorylation patterns of the ion channel TRPM7 which is involved in a range of critical physiological functions. To overcome extraction obstacles associated with analyzing membrane proteins, we incorporated the use of 5% SDS solubilization coupled with SCAD and S-Trap digestion methods to eliminate detergent interference in downstream LC-MS/MS analysis. We found that the SCAD method was more efficient, yielding 84% of the overall identified proteins; however, the variability was greater than the S-Trap method. Using both methods together with TiO 2 and Fe-NTA phospho-enrichment protocols, we successfully observed the phosphorylation pattern of TRPM7 in a transfected cell line. An average of 22 ± 6% of the TRPM7 amino acid sequence was observed. In addition to several previously reported phosphorylation sites, we identified six new phosphosites (S5, S233, S554, S824, T1265, and S1401), providing new targets for further functional analyses of TRPM7.