DirectMS1Quant: Ultrafast Quantitative Proteomics with MS/MS-Free Mass Spectrometry

Recently, we presented the DirectMS1 method of ultrafast
proteome-wide
analysis based on minute-long LC gradients and MS1-only mass spectra
acquisition. Currently, the method provides the depth of human cell
proteome coverage of 2500 proteins at a 1% false discovery rate (FDR)
when using 5 min LC gradients and 7.3 min runtime in total. While
the standard MS/MS approaches provide 4000–5000 protein identifications
within a couple of hours of instrumentation time, we advocate here
that the higher number of identified proteins does not always translate
into better quantitation quality of the proteome analysis. To further
elaborate on this issue, we performed a one-on-one comparison of quantitation
results obtained using DirectMS1 with three popular MS/MS-based quantitation
methods: label-free (LFQ) and tandem mass tag quantitation (TMT),
both based on data-dependent acquisition (DDA) and data-independent
acquisition (DIA). For comparison, we performed a series of proteome-wide
analyses of well-characterized (ground truth) and biologically relevant
samples, including a mix of UPS1 proteins spiked at different concentrations
into an Echerichia coli digest used
as a background and a set of glioblastoma cell lines. MS1-only data
was analyzed using a novel quantitation workflow called DirectMS1Quant
developed in this work. The results obtained in this study demonstrated
comparable quantitation efficiency of 5 min DirectMS1 with both TMT
and DIA methods, yet the latter two utilized a 10–20-fold longer
instrumentation time.