Native MS Analysis of Proteins

All proteins were buffer exchanged into 0.2 M ammonium acetate using Bio-Spin P-6 Size Exclusion Spin Columns (BioRad) as previously described⁶². Native MS was performed using a Q-Exactive HF quadrupole mass spectrometer with Ultra-High Mass Range research modifications (Thermo Fisher Scientific). All proteins were ionized using nano-electrospray ionization in positive mode using 1.1–1.5 kV of spray voltage at 200 °C. Samples were analyzed with a 2000–15,000 m/z range and the resolution was set to 15,000. The trapping gas was set to 5 for all samples and 50 V was applied in the source to aid in desolvation. Data were deconvolved and analyzed using UniDec^{63 (link)}.

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Sacco M.D., Wang S., Adapa S.R., Zhang X., Lewandowski E.M., Gongora M.V., Keramisanou D., Atlas Z.D., Townsend J.A., Gatdula J.R., Morgan R.T., Hammond L.R., Marty M.T., Wang J., Eswara P.J., Gelis I., Jiang R.H., Sun X, & Chen Y. (2022). A unique class of Zn2+-binding serine-based PBPs underlies cephalosporin resistance and sporogenesis in Clostridioides difficile. Nature Communications, 13, 4370.

Publication 2022

Ultra-High Mass Range research modifications

Ammonium acetate Buffer Proteins

Corresponding Organization :

Other organizations : University of South Florida, University of Arizona, Rutgers, The State University of New Jersey

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Variable analysis

independent variables

Ionization method (nano-electrospray ionization in positive mode)
Spray voltage (1.1–1.5 kV)
Source temperature (200 °C)
Mass-to-charge ratio range (2000–15,000 m/z)
Resolution (15,000)
Trapping gas (set to 5)
Source voltage (50 V)

dependent variables

Mass spectrometry data (deconvolved and analyzed using UniDec)

control variables

Buffer exchange of all proteins into 0.2 M ammonium acetate using Bio-Spin P-6 Size Exclusion Spin Columns (BioRad)

positive controls

No positive controls mentioned

negative controls

No negative controls mentioned

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