on 09-20-201902:40 PM - edited on 10-15-202111:30 AM by AnalyteGuru
Sergei Snovida1, Ryan Bomgarden1, Amarjeet Flora1, Bhavin Patel1, Sarah Trusiak2, Emily I. Chen2, John Rogers1, Kay Opperman1
Advances in mass spectrometry (MS) instrumentation has enabled routine analysis of complex protein samples. However, sample preparation methods are not standardized with many protocols taking 8-24 hours in addition to suffering from low peptide yields, poor digestion efficiency and low reproducibility. Here, we describe a simplified sample prep kit containing pre-formulated reagents and a standardized protocol that can be used to efficiently process 10µg to 100µg protein samples in less than 2 hours. In this study, we evaluated the scalability, compatibility, and reproducibility of this sample preparation kit compared to previous published methods. Our new standardized workflow yielded 10-20% higher number of peptides and proteins with lower missed cleavages (<90%) compared to other commercial MS sample prep kits and protein digest standards. We also developed a new 96-well filter plate format to support higher sample processing throughput amenable to the use with automated liquid handling system for sample preparation, TMT labeling, and peptide clean-up. This format showed nearly identical performance in terms of peptide yield, identification rates, alkylation efficiency and digestion efficiency compared to the manual spin column protocol with better reproducibility among replicates.
1Thermo Fisher Scientific, Rockford, IL, USA
2Thermo Fisher Scientific, Cambridge, MA, USA