Evaluation of Protein Identification and Quantification by the diaPASEF Method on timsTOF SCP

Accurate and precise quantification is crucial in modern
proteomics,
particularly in the context of exploring low-amount samples. While
the innovative 4D-data-independent acquisition (DIA) quantitative
proteomics facilitated by timsTOF mass spectrometers gives enhanced
sensitivity and selectivity for protein identification, the diaPASEF
(parallel accumulation-serial fragmentation combined with data-independent
acquisition) parameters have not been systematically optimized, and
a comprehensive evaluation of the quantification is currently lacking.
In this study, we conducted a thorough optimization of key parameters
on a timsTOF SCP instrument, including sample loading amount (50 ng),
ramp/accumulation time (140 ms), isolation window width (20 m/z), and gradient time (60 min). To further
improve the identification of proteins in low-amount samples, we utilized
different column settings and introduced 0.02% n-dodecyl-β-d-maltoside (DDM) in the sample reconstitution solution, resulting
in a remarkable 19-fold increase in protein identification at the
single-cell-equivalent level. Moreover, a comprehensive comparison
of protein quantification using a tandem mass tag reporter (TMT-reporter),
complement TMT ions (TMTc), and diaPASEF revealed a strong correlation
between these methods. Both diaPASEF and TMTc have effectively addressed
the issue of ratio compression, highlighting the diaPASEF method’s
effectiveness in achieving accurate quantification data compared to
TMT reporter quantification. Additionally, an in-depth analysis of
in-group variation positioned diaPASEF between the TMT-reporter and
TMTc methods. Therefore, diaPASEF quantification on the timsTOF SCP
instrument emerges as a precise and accurate methodology for quantitative
proteomics, especially for samples with small amounts.