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The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF)
for Deep Shotgun Proteomics
#MMPMID25991688
Beck S
; Michalski A
; Raether O
; Lubeck M
; Kaspar S
; Goedecke N
; Baessmann C
; Hornburg D
; Meier F
; Paron I
; Kulak NA
; Cox J
; Mann M
Mol Cell Proteomics
2015[Jul]; 14
(7
): 2014-29
PMID25991688
show ga
Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major
principles used in proteomics. Although based on simple fundamentals, it has over
the last decades greatly evolved in terms of achievable resolution, mass
accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes
advantage of these developments and here we develop and evaluate the impact II
for shotgun proteomics applications. Adaption of our heated liquid chromatography
system achieved very narrow peptide elution peaks. The impact II is equipped with
a new collision cell with both axial and radial ion ejection, more than doubling
ion extraction at high tandem MS frequencies. The new reflectron and detector
improve resolving power compared with the previous model up to 80%, i.e. to
40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and
found very high transmission (>80%) up to the collision cell. Simulation and
measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for
QTOF data, improving absolute average mass deviations to better than 1.45 ppm.
More than 4800 proteins can be identified in a single run of HeLa digest in a 90
min gradient. The workflow achieved high technical reproducibility (R2 > 0.99)
and accurate fold change determination in spike-in experiments in complex
mixtures. Using label-free quantification we rapidly quantified haploid against
diploid yeast and characterized overall proteome differences in mouse cell lines
originating from different tissues. Finally, after high pH reversed-phase
fractionation we identified 9515 proteins in a triplicate measurement of HeLa
peptide mixture and 11,257 proteins in single measurements of cerebellum-the
highest proteome coverage reported with a QTOF instrument so far.
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