Mass Spectrometry Acquisition and Fractionation Recommendations for TMT11 and TMT16 Labeled Samples

Proteome coverage and accurate protein
quantification are both
important for evaluating biological systems; however, compromises
between quantification, coverage, and mass spectrometry (MS) resources
are often necessary. Consequently, experimental parameters that impact
coverage and quantification must be adjusted, depending on experimental
goals. Among these parameters is offline prefractionation, which is
utilized in MS-based proteomics to decrease sample complexity resulting
in higher overall proteome coverage upon MS analysis. Prefractionation
leads to increases in required MS analysis time, although this is
often mitigated by isobaric labeling using tandem-mass tags (TMT),
which allow samples to be multiplexed. Here we evaluate common prefractionation
schemes, TMT variants, and MS acquisition methods and their impact
on protein quantification and coverage. Furthermore, we provide recommendations
for experimental design depending on the experimental goals.