Trypsin-induced proteome alteration during cell subculture in mammalian cells.
BACKGROUND
It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions.


METHODS
In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting.


RESULTS
36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization.


CONCLUSIONS
In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.
Huang
Hsiang-Ling
HL
Institute of Bioinformatics and Structural Biology & Department of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan.
Hsing
Hsiang-Wei
HW
Lai
Tzu-Chia
TC
Chen
Yi-Wen
YW
Lee
Tian-Ren
TR
Chan
Hsin-Tsu
HT
Lyu
Ping-Chiang
PC
Wu
Chieh-Lin
CL
Lu
Ying-Chieh
YC
Lin
Szu-Ting
ST
Lin
Cheng-Wen
CW
Lai
Chih-Ho
CH
Chang
Hao-Teng
HT
Chou
Hsiu-Chuan
HC
Chan
Hong-Lin
HL
D001943:Breast Neoplasms / Q000378:metabolism; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005260:Female; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism; D020543:Proteome / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000378:metabolism
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D009363:Neoplasm Proteins; D020543:Proteome; D019253:Proto-Oncogene Proteins c-bcl-2; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D014357:Trypsin
Methods Mol Biol. 2008;424:73-85
18369854
Proteomics. 2003 Oct;3(10):1947-54
14625857
Hum Pathol. 1997 Jun;28(6):734-9
9191009
Proteomics. 2008 Dec;8(23-24):4886-97
19003860
Int J Cancer. 2004 Sep 20;111(5):711-9
15252840
J Biol Chem. 2003 Feb 28;278(9):7607-16
12493773
Free Radic Biol Med. 2010 Jul 1;49(1):96-108
20385227
Cancer Res. 2002 Mar 1;62(5):1541-8
11888933
Proteomics. 2006 Jan;6(2):579-85
16372259
J Biol Chem. 2010 Feb 12;285(7):4307-18
19996095
Anal Bioanal Chem. 2005 Jun;382(3):669-78
15900442
Biochimie. 2002 Feb-Mar;84(2-3):187-93
12022949
Proteomics. 2005 Jul;5(11):2908-26
15954156
J Proteome Res. 2010 Mar 5;9(3):1302-22
20052998
Oncogene. 2006 Jan 12;25(2):248-59
16132038
APMIS. 2005 Oct;113(10):683-7
16309427
FEBS Lett. 2006 May 29;580(13):3229-36
16697377
BMC Cancer. 2006 Dec 06;6:278
17150109