Toward Zero Variance in Proteomics Sample Preparation: Positive-Pressure FASP in 96-Well Format (PF96) Enables Highly Reproducible, Time- and Cost-Efficient Analysis of Sample Cohorts

Abstract

As novel liquid chromatography–mass spectrometry (LC-MS) technologies for proteomics offer a substantial increase in LC-MS runs per day, robust and reproducible sample preparation emerges as a new bottleneck for throughput. We introduce a novel strategy for positive-pressure 96-well filter-aided sample preparation (PF96) on a commercial positive-pressure solid-phase extraction device. PF96 allows for a five-fold increase in throughput in conjunction with extraordinary reproducibility with Pearson product-moment correlations on the protein level of r = 0.9993, as demonstrated for mouse heart tissue lysate in 40 technical replicates. The targeted quantification of 16 peptides in the presence of stable-isotope-labeled reference peptides confirms that PF96 variance is barely assessable against technical variation from nanoLC-MS instrumentation. We further demonstrate that protein loads of 36–60 μg result in optimal peptide recovery, but lower amounts ≥3 μg can also be processed reproducibly. In summary, the reproducibility, simplicity, and economy of time provide PF96 a promising future in biomedical and clinical research.

Quality Control—A Stepchild in Quantitative Proteomics: A Case Study for the Human CSF Proteome

“…Irreproducible carbamylation and digestion could be a reason for the lower-standard quality demonstrated by the MaCProQC tool ( Figure 7 ) and quantitative cluster analysis ( Figure 9 ). If the observed inconsistencies in peptide recovery are related to the filter itself, the reproducibility of the FASP protocol may be improved by the application of an automated positive-pressure approach [ 66 ].…”

Bottom-Up Proteomics: Advancements in Sample Preparation

“…In a recent study, a Resolvex A200 positive-pressure solid-phase extraction unit was combined with a Bravo liquid-handling platform to perform positive-pressure FASP in a 96-well plate [ 111 ]. The method exhibited high reproducibility (Pearson correlation coefficient: r = 0.9993) when analyzing 40 technical replicates of mouse heart tissue lysates.…”

Automated Proteomics Workflows for High-Throughput Library Generation and Biomarker Detection Using Data-Independent Acquisition

“…More recently, Loroch et al introduced a 96-sample format FASP and used positive pressure for buffer exchange steps. Complete automation, however, has not been achieved by the authors because the workflow relied on multiple systems between which the samples had to be moved manually leaving room for further developments in this area . Bead-based sample preparation workflows have recently been introduced offering certain advantages over universal sample preparation based on FASP, and their partial automation has been achieved. , …”

“…Complete automation, however, has not been achieved by the authors because the workflow relied on multiple systems between which the samples had to be moved manually leaving room for further developments in this area. 17 offering certain advantages over universal sample preparation based on FASP, and their partial automation has been achieved. 18,19 Peptide desalting is another universally applied protocol in proteomics investigations.…”