Filter-Based Protein Digestion (FPD): A Detergent-Free and Scaffold-Based Strategy for TMT Workflows

High-throughput
proteome profiling requires thorough optimization
to achieve comprehensive analysis. We developed a filter aided sample
preparation (FASP)-like, detergent-free method, termed Filter-Based
Protein Digestion (FPD). We compared FPD to protein extraction methods
commonly used in isobaric tag-based proteome profiling, namely trichloroacetic
acid (TCA) and chloroform–methanol (C–M) precipitation.
We divided a mammalian whole cell lysate from the SH-SY5Y neuroblastoma
cell line for parallel protein processing with TCA (n = 3), C–M (n = 2), and FPD using either
10 kDa (n = 3) or 30 kDa (n = 3)
molecular weight cutoff membranes. We labeled each sample with tandem
mass tag (TMT) reagents to construct a TMT11-plex experiment. In total,
8654 proteins were quantified across all samples. Pairwise comparisons
showed very little deviation for individual protein abundance measurements
between the two FPD methods, whereas TCA and FPD showed the most difference.
Specifically, membrane proteins were more readily quantified when
samples were processed using TCA precipitation than other methods
tested. However, globally, only 4% of proteins differed greater than
4-fold in the most divergent pair of protein extraction methods (i.e.,
FPD10 and TCA). We conclude that the detergent-free FPD strategy,
particularly using the faster-flowing 30 kDa filter, is a seamless
alteration to high-throughput TMT workflows.