Trypsin-induced proteome alteration during cell subculture in mammalian cells

Abstract

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.

Figure 1

Cell dissociation workflow with and without trypsin digestion. (A) The dissociation time for attached MCF-7 cells were determined where 90% confluent cells in 96-well cell culture plates were gently washed with HBSS twice followed by treated with 100 μl of non-enzymatic cell dissociation solution or 0.05% trypsin-EDTA. After indicated treatment times, cells were gently washed with HBSS and the number of adherent cells counted. The mean cell number of 4 independent assays is shown +/- SD. (B) Overview of cell dissociation workflow with and without trypsin digestion of adhesive MCF-7 cells.

Figure 2

2D-DIGE analysis of the trypsin-induced differentially expressed proteins in MCF-7 cells. Protein samples purified from total cell lysates were labeled with Cy dyes and separated using 24-cm, pH 3-10 nonlinear IPG strip. The differentially expressed protein features are annotated with protein names. The detail information for these identified proteins is listed in Additional file 1.

Figure 3

Representative immunoblotting analysis for selected differentially expressed proteins during trypsinization. The levels of identified proteins (A) tropomyosin alpha-1, (B) HSP-60, (C) SCaMC-1, (D) VDAC2, (E) VDAC1, and (F) CD63 in MCF-7 cells were analyzed by immunoblotting (left top panels), protein expression map from 2D-DIGE (left middle panels), three-dimensional spot images (left bottom panels) and relative quantification of western blotting and 2D-DIGE data for each target protein (right panels).

Figure 4

Expression level analysis of tropomyosin alpha-1, HSP-60, SCaMC-1, VDAC1, VDAC2 and CD63 during trypsinization in Hela cells.

Figure 5

Expression profiles for proteins potentially contributing to (A) metabolism (B) growth regulation (C) electron transportation (D) apoptosis (E) cell adhesion in comparing MCF-7 cells treated with 0.05% trypsin for 10 min followed by recovery for 0 hr, 8 hr and 24 hr or left untreated. The horizontal bars represent fold change in protein expression and the vertical axis indicates the identified proteins. Additional details for each protein can be found in Additional file 1.

Figure 6

Comparison of trypsin-induced cell signaling in cell survival, apoptosis and cell cycle regulation. Activation of survival, apoptosis and cell cycle inhibition signalling pathways were analyzed by immunoblotting with anti-Bcl2, anti-p53 and anti-p21 antibodies, respectively.