In clinical and omics workflows, the quality control sample is relied upon to monitor instrument health and performance. In this work we assess isotopologues of known quantity and spiking them into a human leukemia cell line. We explore low sample concentrations run on short gradients (<30 min) to simultaneously optimize qualitative and quantitative qualification in a short period of time.
Human myelogenous leukemia cell line (K562 - Promega) was reconstituted to 22.5-180 ng/uL where a series of 35 peptide isotopologues (Pierce 7x5) at 5 orders of magnitude different concentrations, between 100 fmol (heaviest) to 8 amol (lightest), were spiked into the cell lysate. Separation was performed on a 25 cm Ionopticks Aurora column Coupled to a timsTOF Pro mass spectrometer (Bruker) operated in PASEF mode. Data analysis was performed with PEAKS Studio (BSI)
To date we tested and optimized experimental conditions to achieve optimal instrument performance, which can be applied to multi-omic and clinical workflows. Peptide digest concentrations (12.5 ng to 180ng), isotopologue loads (8 amol-100 fmol on column), gradient length (8 & 18 min), column length (10 & 25 cm) and tims settings (PASEF scans, cycle time, accumulation and CCS range) were tested to develop a fast, reproducible and robust LC-MS method.
Optimized timsTOF Pro settings are as followed: Capillary voltage was 1600V, gas flow 3 l/min, gas temp: 180C, imex values were between 0.7 and 1.50 V • s/cm2 (End set to 2.10 V • s/cm2), accumulation time was 100 ms, cycle time approximately 73.0 ms (with 100% duty cycle enabled), 10 PASEF MS/MS scans and a total cycle time of 0.87sec.
When ion mobility and CCS merge with the complexity of a digested lysate, spiked with known levels of isotopologues the result is the most complete approach to monitor instrument sensitivity and performance.