Quantitative mass spectrometry: Proteomic analysis of differentiation of MEL cells treated with hexamethylene bisacetamide and 7-[N-(3-aminopropyl)amino] heptan-2-one

• Main Author: Hawke, David H.
• Format: Others
• Published: Scholarly Commons 2004
• Subjects: Biochemistry; Analytical chemistry; Pure sciences; Aminopropyl amino heptan-2-one-7-N-3; Hexamethylene bisacetamide; Proteomics
• Online Access: https://scholarlycommons.pacific.edu/uop_etds/2691; https://scholarlycommons.pacific.edu/cgi/viewcontent.cgi?article=3690&context=uop_etds

Polyamines are small, polycationic molecules required for growth and development and found in all living cells. In this study, the effects of two polyamine analogues, hexamethylene bisacetamide (HMBA), a differentiation inducer, and 7-[N-(3-aminopropyl)amino] heptan-2-one (APAH), an inhibitor of N8-acetylspermidine deacetylase, were studied using quantitative proteomics and stable-isotopes. Two new technologies, isotope-coded affinity tags (ICAT) and quantification in fragment spectra using isobaric stable isotope reagents (iTRAQ) were employed and compared. Quantitative results of these experiments showed few changes in the type and level of proteins detected in whole-cell extracts. Proteins from three populations of cells were studied, control (untreated), HMBA-treated, and HMBA plus APAH treated cells. Some of the proteins that were differentially expressed in response to these agents include pyruvate kinase (PK), lactate dehydrogenase (LDH), mini-chromosome maintenance protein 3 (MCM3), and poly-rC binding protein. The proteins PK and LDH have been reported as possible cancer markers. Histone protein levels were significantly reduced on HMBA treatment, and substantially recovered with the addition of APAH. This finding was very convincing in the iTRAQ work, but invisible to the ICAT experiment, because of the lack of cysteine residues required for quantification in the ICAT methodology. Two proteins were elevated in the HMBA-APAH experiment compared to the other two, heterogeneous nuclear ribonuclear protein C1/C2 (HNRP C1/C2) and ubiquitin. Considering their unique functions, the up-regulation of these proteins suggests the involvement of internal ribosome entry and protein degradation in response to APAH. The results of the two technologies, ICAT and iTRAQ, were found to overlap, but were partly complementary.