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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Project description:This series represents a group of five samples (from sigmoid colon biopsies of clinically normal Patients A-E) profiled by a publicly available cDNA-based platform (GPL284) and a commercial oligonucleotide-based platform (GPL91). Sample RNA extraction: Biopsies taken during endoscopic analysis were immediately snap-frozen in liquid nitrogen and at no time prior to or during subsequent sample manipulation were the samples allowed to thaw. Standard laboratory procedures to eliminate the presence of RNases, including the use of RNase free plastic-ware, heat baked glassware, molecular biology grade chemicals and DEPC-treated solutions, were adopted. Frozen biopsies were crushed to a fine powder under liquid nitrogen using a manual crusher with a teflon head (Omnilab) and total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturers protocol, with minor modifications that improved RNA yields and purity in our hands. In brief, crushed biopsy samples were lysed in an appropriate volume of Buffer RLT (typically 600 µL/20 mg tissue). To facilitate complete lysis, samples were allowed to stand at room temperature for 5-10 min before vortexing. The lysate was homogenized by addition to a QIA shredder column (Qiagen) and centrifuged at 14,000 rpm for 2 min. The resulting supernatant was centrifuged at 14,000 rpm for 3 min to pellet any cell debris. One volume of 70% ethanol was added to the cleared lysate, added to the RNeasy column and centrifuged at 10,000 rpm for 15 sec. Bound RNA was washed with the addition of RW1 buffer to the column and allowed to stand at room temperature for 10 min followed by centrifugation at 10,000 rpm for 15 sec. DNase 1 incubation mix (Stock: 273 kunits; RNase-free DNase Set, Qiagen) was added directly to the spin column membrane and digestion of any contaminating genomic DNA was carried out at room temperature for 30 min. The column was washed with RWI buffer and the DNase treatment step was repeated. The bound RNA was washed with RW1 buffer and centrifuged at 10,000 rpm for 15 sec three times, followed by two additional wash and centrifugation steps with RPE buffer. Finally, purified RNA was eluted off the column with RNase-free water. RNA concentration and purity was determined spectrophotometrically at A260/A280 and was determined to be intact as assessed by formaldehyde gel electrophoresis. The presence of genomic DNA contamination was assessed by PCR amplification of GAPDH using standard PCR conditions and the following primers: GAPDH_F2-5’- ACCCACTCCTCCACCTTTGAC-3’, GAPDH_R2- 5’-CTGTTGCTGTAGCCAAATTCGT-3’. RNA was re-treated with DNase if necessary. Upon confirming the quality of the RNA (intact and free of genomic DNA), 15 µg of total RNA from each of the five biological replicates was used for expression screening on either the Affymetrix or the clone-based filter platform. Keywords: parallel sample

Dataset Information

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

Publications

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

Similar Datasets

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