Improved Peak Detection and Deconvolution of Native Electrospray Mass Spectra from Large Protein Complexes.
Native electrospray-ionization mass spectrometry (native MS) measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact protein assemblies. However, native spectra derived from these assemblies are often partially obscured by low signal-to-noise as well as broad peak shapes because of residual solvation and adduction after the electrospray process. The wide peak widths together with the fact that sequential charge state series from highly charged ions are closely spaced means that native spectra containing multiple species often suffer from high degrees of peak overlap or else contain highly interleaved charge envelopes. This situation presents a challenge for peak detection, correct charge state and charge envelope assignment, and ultimately extraction of the relevant underlying mass values of the noncovalent assemblages being investigated. In this report, we describe a comprehensive algorithm developed for addressing peak detection, peak overlap, and charge state assignment in native mass spectra, called PeakSeeker. Overlapped peaks are detected by examination of the second derivative of the raw mass spectrum. Charge state distributions of the molecular species are determined by fitting linear combinations of charge envelopes to the overall experimental mass spectrum. This software is capable of deconvoluting heterogeneous, complex, and noisy native mass spectra of large protein assemblies as demonstrated by analysis of (1) synthetic mononucleosomes containing severely overlapping peaks, (2) an RNA polymerase II/α-amanitin complex with many closely interleaved ion signals, and (3) human TriC complex containing high levels of background noise. Graphical Abstract ᅟ.
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA.|Princeton University, Princeton, NJ, 08544, USA.
Jonathan
J
Lu
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA.
Michael J
MJ
Trnka
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, 77030, USA.
Soung-Hun
SH
Roh
Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, 94305, USA.
Philip J J
PJ
Robinson
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA.|Chemistry and Chemical Biology Graduate Program, University of California, San Francisco, CA, 94158, USA.
Carrie
C
Shiau
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA.|Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94158, USA.
Danica Galonic
DG
Fujimori
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, 77030, USA.
Wah
W
Chiu
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA.
Alma L
AL
Burlingame
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA. sguan@cgl.ucsf.edu.|Institute for Neurodegenerative Diseases, University of California, San Francisco, CA, 94143, USA. sguan@cgl.ucsf.edu.
Shenheng
S
Guan
D000465:Algorithms; D053959:Alpha-Amanitin / Q000378:metabolism; D000818:Animals; D056404:Chaperonin Containing TCP-1 / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009707:Nucleosomes / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014981:Xenopus / Q000378:metabolism; D029867:Xenopus Proteins / Q000378:metabolism
D053959:Alpha-Amanitin; D006657:Histones; D009707:Nucleosomes; D011506:Proteins; D029867:Xenopus Proteins; D012319:RNA Polymerase II; D056404:Chaperonin Containing TCP-1
Deconvolution; Native mass spectrometry; Protein assemblies; Protein complexes