SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence

<p>Abstract</p> <p>Background</p> <p>The adaptive immune response requires waves of T-cell clonal expansion on contact with pathogen and elimination after clearance of the source of antigen. However, lifelong persistent infections with common viruses cause chronic antigenic stimulation which takes its toll on adaptive immunity in late life. Chronic antigenic stress results in deregulation of the T-cell response and accumulation of anergic cells. Longitudinal studies of the elderly show that this impacts on survival. Identifying the nature of the defects in chronically-stimulated T-cells and protein bio-markers of these dysfunctional cells would help to understand age-associated compromised T-cell function (immunosenescence) and facilitate the development of targeted intervention strategies.</p> <p>The purpose of this work was to use surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to analyse proteins associated with T-cell senescence in order to identify potential bio-markers. Clonal populations of T-cells isolated from elderly octogenarian and centenarian donors were grown <it>in vitro </it>until senescence, and early passage and late passage (pre-senescent) cells were analysed using SELDI-TOF-MS ProteinChip arrays.</p> <p>Results</p> <p>Discriminant analysis identified several protein or peptide peaks in the region of 14.5–16.5 kDa that were associated with T-cell clone senescence. Human profilin-1, a ubiquitous protein associated with actin remodelling and cellular motility was unambiguously identified. Altered expression of profilin-1 in senescent T-cell clones was confirmed by Western blot analysis.</p> <p>Conclusion</p> <p>Due to the proposed roles of profilin-1 in cellular survival, cytoskeleton remodelling, motility, and proliferation, it is hypothesised that differential expression of profilin-1 in ageing may contribute directly to immunosenescence.</p

SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence-1

<p><b>Copyright information:</b></p><p>Taken from "SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence"</p><p>http://www.proteomesci.com/content/5/1/7</p><p>Proteome Science 2007;5():7-7.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892543.</p><p></p>p analysis. A region between 14.5 kDa and 16.5 kDa, marked by asterisk, was identified to be highly conserved between individual samples and significantly differed between early and late passage T-cell clones

SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence-7

<p><b>Copyright information:</b></p><p>Taken from "SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence"</p><p>http://www.proteomesci.com/content/5/1/7</p><p>Proteome Science 2007;5():7-7.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892543.</p><p></p>p analysis. A region between 14.5 kDa and 16.5 kDa, marked by asterisk, was identified to be highly conserved between individual samples and significantly differed between early and late passage T-cell clones

SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence-5

<p><b>Copyright information:</b></p><p>Taken from "SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence"</p><p>http://www.proteomesci.com/content/5/1/7</p><p>Proteome Science 2007;5():7-7.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892543.</p><p></p>d lysed in sample buffer (50 mM Tris, pH 8.0, 120 mM NaCl, 0.5% NP40, 10 μg/mL PMSF (phenylmethylsulfonylfluoride), and 1× protease inhibitor cocktail. A total of 30 μg protein was suspended in laemmli buffer (50 mM tris, pH 6.8, 1% B-mercaptoethanol, 2% sodium dodecyl sulphate, 0.1% bromophenol blue, and 10% glycerol). Samples were boiled and subject to Western blot analysis on NuPAGE™ Bis-Tris gels with MES running buffer. Human profilin-1 and β-actin proteins were detected and visualised by enhanced chemi-luminescence. Protein expression profiles from one representative clone from an octogenarian and centenarian donor is shown along with population doublings (PD) for each sample analysed (A). Average profilin-1 expression in early and late passage octogenarian and centenarian samples is depicted graphically following normalisation to β-actin expression in each sample (B). Student's t-test was performed and p < 0.05 was considered statistically significant

SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence-8

<p><b>Copyright information:</b></p><p>Taken from "SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence"</p><p>http://www.proteomesci.com/content/5/1/7</p><p>Proteome Science 2007;5():7-7.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892543.</p><p></p>in triplicate. From the list of peak clusters created between 14.5 kDa and 16.5 kDa m/z, p-values were calculated using CiphergenExpress Client software (version 3.0.5.013). Peaks were manually scored for quality and peaks with signal:noise ratios (S/N

SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence-3

<p><b>Copyright information:</b></p><p>Taken from "SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence"</p><p>http://www.proteomesci.com/content/5/1/7</p><p>Proteome Science 2007;5():7-7.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892543.</p><p></p>igh-performance liquid chromatography (HPLC) was performed in order to compare mass obtained from SELDI analysis to more accurate MALDI-TOF MS. HPLC-purified samples were run in parallel on SELDI-TOF (on a normal-phase ProteinChip) and MALDI-TOF mass spectrometers. One peak of 14.9 kDa identified as differentially expressed in T-cell senescence is demonstrated by MALDI-TOF (a) and SELDI-TOF (b) mass spectrometric analysis

SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence-4

<p><b>Copyright information:</b></p><p>Taken from "SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence"</p><p>http://www.proteomesci.com/content/5/1/7</p><p>Proteome Science 2007;5():7-7.</p><p>Published online 5 Jun 2007</p><p>PMCID:PMC1892543.</p><p></p>nylfluoride), and 1× protease inhibitor cocktail. A total of 30 μg protein was suspended in laemmli buffer (50 mM tris, pH 6.8, 1% β-mercaptoethanol, 2% sodium dodecyl sulphate, 0.1% bromophenol blue, and 10% glycerol). Samples were boiled and subject to Western blot analysis on NuPAGE™ Bis-Tris gels with MES running buffer. Human profilin-1, cofilin and β-actin proteins were detected by antibodies as detailed in and visualised by enhanced chemi-luminescence