Dataset Information

Spin column-based prefractionation alternatives for Streamlined Tandem Mass Tag (SL-TMT) sample processing

ABSTRACT: Fractionation is essential to achieving deep proteome coverage for sample multiplexing experiments where currently up to 18 samples can be analyzed concurrently. However, prefractionation (i.e., upstream of LC-MS/MS analysis) with a liquid chromatography system constrains sample processing as only a single sample can be fractionated at once. Here, we highlight the use of spin column-based methods which permit multiple multiplexed samples to be prefractionated simultaneously. These methods require only a centrifuge and eliminate the need for a dedicated liquid chromatograph. We investigate prefractionation with strong anion exchange (SAX) and high-pH reversed phase (HPRP) spin columns, as well as a combination of both, as methods of prefractionation. In two separate experiments, we acquired deep proteome coverage (>8,000 quantified proteins), while starting with only 25 µg of protein per channel. Our datasets showcase the proteome alterations in two human cell lines resulting from treatment with inhibitors acting on the ubiquitin-proteasome system. We recommend this spin column-based prefractionation strategy for high-throughput screening applications or whenever a liquid chromatograph is not readily available.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Joao Paulo

LAB HEAD: Joao A. Paulo

PROVIDER: PXD038383 | Pride | 2023-12-28

REPOSITORIES: Pride

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Publications

Spin column-based peptide fractionation alternatives for streamlined tandem mass tag (SL-TMT) sample processing.

Liu Xinyue X Rossio Valentina V Paulo Joao A JA

Journal of proteomics 20230208

Fractionation is essential to achieving deep proteome coverage for sample multiplexing experiments where currently up to 18 samples can be analyzed concurrently. However, peptide fractionation (i.e., upstream of LC-MS/MS analysis) with a liquid chromatography system constrains sample processing as only a single sample can be fractionated at once. Here, we highlight the use of spin column-based methods which permit multiple multiplexed samples to be fractionated simultaneously. These methods requ ...[more]

PMID: 36758854

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