Two dimensional gel phosphoproteome of peripheral blood mononuclear cells: comparison between two enrichment methods

Proteome Science

Research Article

Loreto Gesualdo¹  Maria Teresa Rocchetti²  Leonarda Varraso³  Giuseppe Grandaliano³  Michela Alfarano⁴  Elena Ranieri⁵  Massimo Papale⁵  Salvatore Di Paolo⁶ 

[1] Department of Emergency and Organ Transplantation (DETO), Nephrology, Dialysis and Transplantation Unit, University of Bari Aldo Moro, Piazza G. Cesare, 11, 70124, Bari, Italy;Department of Emergency and Organ Transplantation (DETO), Nephrology, Dialysis and Transplantation Unit, University of Bari Aldo Moro, Piazza G. Cesare, 11, 70124, Bari, Italy;Department of Medical and Surgical Sciences, Proteomics and Mass Spectrometry Core Facility, University of Foggia, Foggia, Italy;Department of Medical and Surgical Sciences, Nephrology, Dialysis and Transplantation Unit, University of Foggia, Foggia, Italy;Department of Medical and Surgical Sciences, Proteomics and Mass Spectrometry Core Facility, University of Foggia, Foggia, Italy;Department of Medical and Surgical Sciences, Proteomics and Mass Spectrometry Core Facility, University of Foggia, Foggia, Italy;Clinical Pathology Unit, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy;Nephrology and Dialysis Unit, Hospital Dimiccoli, Barletta, Italy;

关键词: 2DE;    Lanthanum;    PBMCs;    Phosphoproteome;    Phosphoprotein enrichment;   

DOI  :  10.1186/s12953-014-0046-1

 received in 2014-05-12, accepted in 2014-08-14,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundProtein phosphorylation is considered a key event in signal transduction. Peripheral blood mononuclear cells (PBMCs) are a critical component of the immune system. The analysis of PBMCs phosphoproteome might help elucidate the signaling pathways essential to their biological role in health, immunological diseases and cancer. Enrichment of phosphoproteins becomes a prerequisite for phosphoproteome analysis and conventionally requires a multi-step procedure and sophisticated equipments. In this study, we standardized 2D-PAGE phosphoproteome analysis of PBMCs and compared two phosphoprotein enrichment methods, lanthanum chloride precipitation and affinity micro-column. Further, the different specificity for PBMCs phosphorylated proteins of each method was investigated.ResultsPBMCs were isolated from fresh whole blood of ten healthy donors. PBMCs phosphoproteins were enriched either by phosphoprotein precipitation using lanthanum chloride, with an overall yield of 8.9 ± 4.7%, or by using an affinity micro-column, with a lower yield of 3.2 ± 1.6% (p = 0.05). Image analysis of Sypro-stained analytical 2D-PAGE gels detected 554 ± 68 protein spots for the lanthanum pattern [inter-assay coefficient of variation (CV) = 27.0%, intra-assay CV = 10.7%] and 575 ± 35 protein spots for the micro-column pattern (inter-assay CV = 26.8%; intra-assay CV = 11.0%) (p = 0.6), with 57% match of the spots detected by the 2 approaches. 1D gel electrophoresis and western blot analyses of the supernatants suggested a better lanthanum ions capability to deplete phosphoproteins in a PBMCs protein lysate compared to the affinity micro-column. On the other hand, 1D gel electrophoresis analysis of dephosphorylated PBMCs protein lysate revealed a relatively higher unspecificity for the lanthanum ions compared to affinity micro-column. Filamin-A, coronin 1A, pyruvate kinase isozymes M1/M2 and ficolin-1 were considerably more expressed in the lanthanum phosphoprotein pattern. Interestingly, ficolin-1 had not been reported in 2DE-PBMCs proteome profiles so far.ConclusionOur results describe the differences and the validity of phosphoprotein enrichment methods and provide two successful and complementary approaches for the 2DE phosphoproteome analysis of PBMCs.

【 授权许可】

Unknown   
© Rocchetti et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.