Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

In-depth TMT Quantification of native CSF by Tandem mass Tag (TMT) channel boosting. (#504)

Lingyan Ping 1 , DUC M DUONG 1 , James J Lah 1 , Allan I Levey 1 , Nicholas T Seyfried 1

Recent developments in quantitative proteomics has enabled deep and comprehensive analysis of cultured cells and tissues samples.  In combination with isobaric tandem mass tags (TMT) and technical advancements in mass spectrometry, proteome coverage beyond 10K proteins in these samples is readily accomplished.  However, these pipelines and depths have not been easily transferable to biofluids such as cerebrospinal fluid (CSF).  The foremost challenge of profiling CSF is that proteins can span up to 10 orders difference.  In CSF, the top 14 proteins make up more than 80% of the total protein abundance.  Many studies have used immunodepletion approaches to remove these proteins in an effort to achieve better depth and higher coverage of lower abundant species.  These methods however add additional variability due to differences in depletion efficiency and off target depletion of interacting proteins.  Other studies have utilized native non-depleted CSF in conjunction with extensive gel and/or off-line fractionation (up to 40 fractions) to achieve quantification depths beyond 2000 proteins.  This however, involves an unsurmountable amount of preparation and machine time and is not practical for larger scale studies.  Here, we present an efficient and innovative approach that involves both immunodepletion and off-line fractionation to achieve superior results.  With this new approach, we are able to sequence native nondepleted CSF to a depth of > 3000 proteins utilizing less than 3 days of machine time.  Our approach utilizes a “boosted” TMT channel comprised of depleted CSF.  All other TMT channels are comprised of non-depleted CSF samples.  We have applied this optimized pipeline to assess differences in 96 samples from an early stage Alzheimer’s study (32 controls, 32 asymptomatic and 32 cognitively impaired).