title: Two dimensional gel phosphoproteome of peripheral blood mononuclear cells: comparison between two enrichment methods.
abstract: BACKGROUND
  Protein 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.
  
  
  RESULTS
  PBMCs 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Department of Emergency and Organ Transplantation (DETO), Nephrology, Dialysis and Transplantation Unit, University of Bari Aldo Moro, Piazza G. Cesare, 11, Bari, 70124 Italy ; Department of Medical and Surgical Sciences, Proteomics and Mass Spectrometry Core Facility, University of Foggia, Foggia, Italy.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Rocchetti
authors.affiliation: Department of Medical and Surgical Sciences, Proteomics and Mass Spectrometry Core Facility, University of Foggia, Foggia, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Alfarano
authors.affiliation: Department of Medical and Surgical Sciences, Nephrology, Dialysis and Transplantation Unit, University of Foggia, Foggia, Italy.
authors.forename: Leonarda
authors.initials: L
authors.lastname: Varraso
authors.affiliation: Nephrology and Dialysis Unit, Hospital Dimiccoli, Barletta, Italy.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Di Paolo
authors.affiliation: 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.
authors.forename: Massimo
authors.initials: M
authors.lastname: Papale
authors.affiliation: 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.
authors.forename: Elena
authors.initials: E
authors.lastname: Ranieri
authors.affiliation: Department of Medical and Surgical Sciences, Nephrology, Dialysis and Transplantation Unit, University of Foggia, Foggia, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Grandaliano
authors.affiliation: Department of Emergency and Organ Transplantation (DETO), Nephrology, Dialysis and Transplantation Unit, University of Bari Aldo Moro, Piazza G. Cesare, 11, Bari, 70124 Italy.
authors.forename: Loreto
authors.initials: L
authors.lastname: Gesualdo
keywords: 2DE; Lanthanum; PBMCs; Phosphoprotein enrichment; Phosphoproteome