Advancing Top-down Analysis of the Human Proteome Using a Benchtop Quadrupole-Orbitrap Mass Spectrometer.
Over the past decade, developments in high resolution mass spectrometry have enabled the high throughput analysis of intact proteins from complex proteomes, leading to the identification of thousands of proteoforms. Several previous reports on top-down proteomics (TDP) relied on hybrid ion trap-Fourier transform mass spectrometers combined with data-dependent acquisition strategies. To further reduce TDP to practice, we use a quadrupole-Orbitrap instrument coupled with software for proteoform-dependent data acquisition to identify and characterize nearly 2000 proteoforms at a 1% false discovery rate from human fibroblasts. By combining a 3 m/z isolation window with short transients to improve specificity and signal-to-noise for proteoforms >30 kDa, we demonstrate improving proteome coverage by capturing 439 proteoforms in the 30-60 kDa range. Three different data acquisition strategies were compared and resulted in the identification of many proteoforms not observed in replicate data-dependent experiments. Notably, the data set is reported with updated metrics and tools including a new viewer and assignment of permanent proteoform record identifiers for inclusion of highly characterized proteoforms (i.e., those with C-scores >40) in a repository curated by the Consortium for Top-Down Proteomics.
Departments of Chemistry and Molecular Biosciences, Northwestern University , 2170 Campus Drive, Evanston, Illinois 60208, United States.
Luca
L
Fornelli
Departments of Chemistry and Molecular Biosciences, Northwestern University , 2170 Campus Drive, Evanston, Illinois 60208, United States.
Kenneth R
KR
Durbin
Departments of Chemistry and Molecular Biosciences, Northwestern University , 2170 Campus Drive, Evanston, Illinois 60208, United States.
Ryan T
RT
Fellers
Departments of Chemistry and Molecular Biosciences, Northwestern University , 2170 Campus Drive, Evanston, Illinois 60208, United States.
Bryan P
BP
Early
Departments of Chemistry and Molecular Biosciences, Northwestern University , 2170 Campus Drive, Evanston, Illinois 60208, United States.
Joseph B
JB
Greer
Departments of Chemistry and Molecular Biosciences, Northwestern University , 2170 Campus Drive, Evanston, Illinois 60208, United States.
Richard D
RD
LeDuc
Departments of Chemistry and Molecular Biosciences, Northwestern University , 2170 Campus Drive, Evanston, Illinois 60208, United States.
Philip D
PD
Compton
Departments of Chemistry and Molecular Biosciences, Northwestern University , 2170 Campus Drive, Evanston, Illinois 60208, United States.
Neil L
0000-0002-8815-3372
NL
Kelleher
D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D020543:Proteome / Q000235:genetics* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012984:Software
D020543:Proteome
AUTOPILOT; Orbitrap; data-dependent acquisition; false-discovery rate; gas-phase fractionation; mass spectrometry; medium/high; proteoform; quadrupole; top-down proteomics