Quantitative Global and Phospho-proteomics analysis of rapamycin-treated human SU-DHL-4 cells by LC-MSMS

ABSTRACT: Global protein profiling and global phospho-protein profiling in human diffuse large B cell lymphoma cell line SU-DHL-4 treated with Rapamycin at two time points compared with DMSO control was performed by Poochon Scientific using the TMT-18plex labeling based quantitative proteomics method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nine frozen cell pellet samples (6 million cells each) were received and processed for the study by Poochon Scientific. The preparation of the protein lysates from 9 frozen pellet samples was performed according to the method described in method section. Protein concentrations were measured using the BCA method. An equal amount of total protein from each sample was processed for MS analysis. In summary, 200 µg of protein from each of the 9 samples was processed for trypsin digestion, followed by TMT-multiplex labeling using one TMT-18plex sets. 10 unique TMT tags were used to label 150 µg of trypsin-digested peptides from each of the 9 digests and one master mixture (an equal amount mixture of 9 digests) (Table 1). After fractionation by UHPLC, 90% of the labeled peptides fractions (6 fractions) was used for enrichment of phospho-peptides, and 10% of the fractions (12 fractions) were analyzed by 12x 110 min LC/MS/MS runs. 10 unique TMT tags from the TMT-18plex set were used to label 9 trypsin-digested peptides plus one master mixture (Table 1). Table 1: Description (Label, BCA (mg/ml), TMT-tag); DMSO rep1 (DMSO1, 4.3 mg/ml, 126);DMSO rep2 (DMSO2, 4.12 mg/ml, 127N);DMSO rep3 (DMSO3, 4.5 mg/ml, 127C);Rapamycin 24h rep1 (Rapamycin24h1, 3.32 mg/ml, 128N);Rapamycin 24h rep2 (Rapamycin24h2, 4.62 mg/ml, 128C);Rapamycin 24h rep3 (Rapamycin24h3, 3.18 mg/ml, 129N);Rapamycin 48h rep1 (Rapamycin48h1, 3.46 mg/ml, 129C);Rapamycin 48h rep2 (Rapamycin48h2, 3.24 mg/ml, 130N);Rapamycin 48h rep3 (Rapamycin48h3, 3.68 mg/ml, 130C);Master mix mix of equal amount of 9 samples (131N). After TMT labeling, the 10 labeled peptides were mixed followed by two fractionations using basic reversed-phase UHPLC. 12 fractions from 10% aliquot were generated and analyzed sequentially by 12x 110-minute LC-MS/MS runs. 12 fractions from 90% aliquot were combined into 6 fractions and then phospho-peptides were enriched by using ThermoFisher TiO2 Phosphopeptide Enrichment Kit and analyzed sequentially by 6x 110-minute LC-MS/MS runs.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): B Cell, Cell Suspension Culture

DISEASE(S): Lymphoma

SUBMITTER: Jens Kalchschmidt  

LAB HEAD: John J

PROVIDER: PXD067812 | Pride | 2025-09-03

REPOSITORIES: Pride