Fig. 2.

Fragmentation behavior for dda-, dia- and synchro-PASEF.A, comparing fragment signals of dda-, dia- and synchro-PASEF. A tryptic digest of HeLa cells was measured in a 21 min gradient. The same frame numbers as in Figure 1B are shown but collision energy was applied. Left panel, fragmentation spectra of 15 precursors marked with their isolation windows in dda-PASEF. Middle panel, isolation windows and continuous fragment acquisition in dia-PASEF. There are no fragments at the transition point and at the precursor low mobility regions. Right panel, the isolation window of the third synchro-PASEF scan is shown. Note the uniform fragment distribution in the entire m/z – ion mobility plane. B, conversion efficiency to fragments for the three scan modes illustrated with the fragment intensities of GLAGVENVTELK (phosphorylase b) and SISIVGSYVGNR (ADH) co-eluting at a retention time of 10.35 to 10.55 min and an ion mobility of 0.93 to 0.955 V cm⁻². In DDA mode, the eluting peptides were fragmented three times in this low complexity mixture. Synchro-PASEF sampled the peptide many more times than dia-PASEF due to shorter cycle times. C, summed intensities for the fragment signals of the two peptides in (B). ADH, alcohol dehydrogenase; DDA, data-dependent acquisition; DIA, data-independent acquisition; PASEF, parallel accumulation—serial fragmentation.