The analysis of large sample cohorts requires regular QC injections to monitor the instrument’s performance over the entirety of the experiment. As throughput concerns grow with the size of the sample cohort, we have developed a QC method that relies on the additional separation power and high sequencing speed of Parallel Accumulation Serial Fragmentation (PASEF) to deliver reproducible and quantitative information from a high-complexity QC sample despite a short injection to injection time.
HeLa peptide digest was dissolved to 250 ng/ µL, where 1 µL injection volume was delivered to a 100 mm fritted column of ID 75 µm (Bruker). The nano LC business logic was modified in a way that turnover times were reduced to achieve a 21.4 min gradient time and 28.8 min inject to inject time. Separation was performed at 500 nl/ min and loading was performed at a max pressure of 500 bar. This method allows up to 50 samples per day to be measured, maximizing throughput. PEAKS studio (Bioinformatics Solutions Inc.) was used for data processing and results were corrected to 1% PSM FDR.
Using the optimized 28.8 min method, more than 4000 protein groups could be identified from 250 ng of a proteolytic digest of a human cancer cell line (HeLa). In parallel, offline LC-fractionation was performed on a HeLa digest and digests of murine cerebellum, and the fractions analyzed using the same column and method. Samples were fractionated on high pH reversed-phase columns into 24 concatenated samples. Subsequent measurement allowed the identification of more than 100000 unique peptides and 9052 protein groups in less than 12 h of measurement time.
This method allows to perform rapid QC from complex samples and expedite the assembly of large spectral libraries from fractionated samples.