Typical challenges in non-targeted protein quantitation are the cycle time (defining the chromatographic peak resolution), the uniqueness of the ion(s) chosen to determine a peptide amount (selectivity) and the system sensitivity (quantitation of low-abundance compounds). MS-based approaches provide comprehensive quantitative results but overlapping ions reduce the selectivity of these workflows. MS/MS (DIA) based quantitation provides excellent selectivity at the cost of reduced sensitivity.
TIMS-PASEF based label-free quantitation (PASEF-LFQ) combines the best of both approaches. TIMS provides both an additional dimension of separation, greatly increasing selectivity, while the preconcentration effect of the TIMS cell provides greater sensitivity. The PASEF cycle time is compatible with narrow nano-UHPLC peaks while its speed (>100 Hz MS/MS) greatly improves the reproducibility of parent ion selection compared to traditional DDA approaches.
In this case, the timsTOF Pro’s unique 4D-MBR approach allows to overcome the missing value problem with an unprecedented sensitivity.
Label-free quantification outcome of a three-proteome mixture. The total load is 150 ng, separated with a 60 min gradient. PeaksX is used for processing, Perseus for display.