Fetal calf serum heat inactivation and lipopolysaccharide contamination influence the human T lymphoblast proteome and phosphoproteome

Abstract

Background: The effects of fetal calf serum (FCS) heat inactivation and bacterial lipopolysaccharide (LPS) contamination on cell physiology have been studied, but their effect on the proteome of cultured cells has yet to be described. This study was undertaken to investigate the effects of heat inactivation of FCS and LPS contamination on the human T lymphoblast proteome. Human T lymphoblastic leukaemia (CCRF-CEM) cells were grown in FCS, either non-heated, or heat inactivated, having low (< 1 EU/mL) or regular (< 30 EU/mL) LPS concentrations. Protein lysates were resolved by 2-DE followed by phospho-specific and silver nitrate staining. Differentially regulated spots were identified by nano LC ESI Q-TOF MS/MS analysis.

Results: A total of four proteins (EIF3M, PRS7, PSB4, and SNAPA) were up-regulated when CCRF-CEM cells were grown in media supplemented with heat inactivated FCS (HE) as compared to cells grown in media with non-heated FCS (NHE). Six proteins (TCPD, ACTA, NACA, TCTP, ACTB, and ICLN) displayed a differential phosphorylation pattern between the NHE and HE groups. Compared to the low concentration LPS group, regular levels of LPS resulted in the up-regulation of three proteins (SYBF, QCR1, and SUCB1).

Conclusion: The present study provides new information regarding the effect of FCS heat inactivation and change in FCS-LPS concentration on cellular protein expression, and post-translational modification in human T lymphoblasts. Both heat inactivation and LPS contamination of FCS were shown to modulate the expression and phosphorylation of proteins involved in basic cellular functions, such as protein synthesis, cytoskeleton stability, oxidative stress regulation and apoptosis. Hence, the study emphasizes the need to consider both heat inactivation and LPS contamination of FCS as factors that can influence the T lymphoblast proteome.

Figure 1

Silver nitrate stained 2-DE gel. (A) Proteins (160 μg) were separated in the first dimension using non-linear pH 3-10 gradient IPG strips (17 cm, Bio-Rad), followed by second dimension on 12.5% SDS-PAGE. Consistently regulated spots were excised from silver stained gel after densitometric analysis for identification by Q-TOF MS/MS analysis. Spots marked on the gel showed differentially regulated proteins. Note: "P" refers to phospho protein spots also shown in figure 2. (B) Two representative differentially regulated 2-DE spots (MOBKL1A, spot 8; SNAPA, spot 10) in non-heated FCS with low LPS (NHL) and in heated FCS with normal LPS concentration (HE) respectively. The spot IDs correspond to the listing in Table 1. The error bars represent mean ± SD (*= p < 0.05, **= p < 0.005) of six independent experiments.

Figure 2

Phospho-specific florescence stained 2-DE. (A) Proteins were resolved on 2-DE and gels were stained by Pro-Q Diamond phospho-stain (Invitrogen) and then scanned (FLA -5100). The spots showing significant regulation after densitometry analysis were marked and identified by Q-TOF MS/MS analysis. (B) Illustration of two representative 2-DE spots (TCTP, spot 16; ACTB; spot 17) in non-heated FCS with normal LPS (NHE) and in heated FCS with normal LPS (HE). The error bars represent mean ± SD (*= p < 0.05) of six independent experiments.

Figure 3

Immunoblot analysis of superoxide dismutase 2 (SOD2) expression. CCRF-CEM lysate treated with non-heated FCS with low LPS (NHL) and in heated FCS with normal LPS (HE), were resolved on 1DE and immunoblotted with antibody against SOD2. Densitometric analyses were done using Lab Image version 2.71 software. β-tubulin was used as a loading control. The error bars represent mean ± SD (*= p < 0.05) of three independent experiments.