Semi-automated positive pressure FASP in 96-well format (PF96) enables highly reproducible and time-efficient proteomics sample preparation of larger cohorts

ABSTRACT: As novel technologies allow for a substantial increase in the number of LC-MS run per day, proteomics sample preparation appears as new bottle-neck for throughput. To overcome this limitation, we introduce a novel strategy for positive pressure 96-well filter-aided sample preparation (PF96) on a commercial positive pressure solid-phase extraction unit allowing for a 5-fold reduction in lab-time. Similar as conventional FASP, PF96 allows for robust processing of protein amounts between 3 and 60 µg, with higher analyte recovery observed for higher protein loads (36-60 µg). Notably, even lower amounts can be processed reproducibly, but at the expense of loss of peptide signal intensity (~40 % of signal intensity for 3 µg compared to 60 µg ) - more pronounced for peptides of higher retention time (reversed phase) and higher share of the hydrophobic amino acids F, L, M, W. Processing of 40 technical replicates of mouse heart tissue lysate highlights its reproducibility with Pearson Product-Moment correlations of r=0.9992 and r=0.9891 on protein and peptide level, respectively, which is similar to the reproducibility of LC-MS for replicate injections of identical samples.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Heart

SUBMITTER: Stefan Loroch  

LAB HEAD: Albert Sickmann

PROVIDER: PXD024236 | Pride | 2021-12-03

REPOSITORIES: Pride