Fig. 3

Hybrid immunoprecipitation HR-PRM-MS method variability. Using two methodological workflows (Fig. 2) the variability in the IP-HR-PRM-MS method targeting LAMP2 peptides was evaluated. Workflow 1 reflects the overall variability of the method whereas Workflow 2 isolated the variation in the micro-LC HR-PRM-MS analysis. Workflow 1 and 2 each included samples prepared on three separate occasions, Batch 1–3, each including 8 + 8 technical replicates. The different batches were analyzed on a single occasion to minimize the influence of altering instrumental performance. The workflow enabled determination of intra- and inter-day coefficients of variations (CVs), where the intraday variation was calculated for each batch and the inter-day variation calculated for the samples included in all three batches. The intra- and inter-day CVs for the workflows are shown for the LAMP2 peptides; a aa 133–144, b aa 145–152, c aa 153–161; and d aa 334–351. The bar graphs show the calculated ratio between the sum of the included fragment ion peak areas of the tryptic peptide against the sum of peak areas of the added isotope labeled peptide for each sample and within each batch sorted, from left to right, in order of analysis