Bioinformatics Analysis of Top-Down Mass Spectrometry Data with ProSight Lite.
Traditional bottom-up mass spectrometry-based proteomics relies on the use of an enzyme, often trypsin, to generate small peptides (typically < 25 amino acids long). In top-down proteomics, proteins remain intact and are directly measured within the mass spectrometer. This technique, while inherently simpler than bottom-up proteomics, generates data which must be processed and analyzed using software tools "purpose-built" for the job. In this chapter, we will show the analysis of intact protein spectra through deconvolution, deisotoping, and searching with ProSight Lite, a free, vendor-agnostic tool for the analysis of top-down mass spectrometry data. We will illustrate with two examples of intact protein fragmentation spectra and discuss the iterative use of the software to characterize proteoforms and discover the sites of post-translational modifications.
Departments of Chemistry, Molecular Biosciences, the Proteomics Center of Excellence and the Robert H. Lurie Comprehensive Cancer Center at the Feinberg School of Medicine, Northwestern University, Evanston, IL, 60208, USA.
Caroline J
CJ
DeHart
Departments of Chemistry, Molecular Biosciences, the Proteomics Center of Excellence and the Robert H. Lurie Comprehensive Cancer Center at the Feinberg School of Medicine, Northwestern University, Evanston, IL, 60208, USA.
Ryan T
RT
Fellers
Departments of Chemistry, Molecular Biosciences, the Proteomics Center of Excellence and the Robert H. Lurie Comprehensive Cancer Center at the Feinberg School of Medicine, Northwestern University, Evanston, IL, 60208, USA.
Luca
L
Fornelli
Departments of Chemistry, Molecular Biosciences, the Proteomics Center of Excellence and the Robert H. Lurie Comprehensive Cancer Center at the Feinberg School of Medicine, Northwestern University, Evanston, IL, 60208, USA.
Neil L
NL
Kelleher
Departments of Chemistry, Molecular Biosciences, the Proteomics Center of Excellence and the Robert H. Lurie Comprehensive Cancer Center at the Feinberg School of Medicine, Northwestern University, Evanston, IL, 60208, USA. paul-thomas@northwestern.edu.
Paul M
PM
Thomas
D000818:Animals; D019295:Computational Biology / Q000379:methods*; D057225:Data Mining / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D012984:Software; D014584:User-Computer Interface; D064878:Web Browser
D011506:Proteins
Bioinformatics; Intact protein; Mass spectrometry; ProSight Lite; Proteomics; Top-down