Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology

Abstract

Systematic parallel analysis of the phosphorylation status of networks of interacting proteins involved in the regulatory circuitry of cells and tissues is certain to drive research in the post-genomics era for many years to come. Reversible protein phosphorylation plays a critical regulatory role in a multitude of cellular processes, including alterations in signal transduction pathways related to oncogene and tumor suppressor gene products in cancer. While fluorescence detection methods are likely to offer the best solution to global protein quantitation in proteomics, to date, there has been no satisfactory method for the specific and reversible fluorescent detection of gel-separated phosphoproteins from complex samples. The newly developed Pro-Q Diamond phosphoprotein dye technology is suitable for the fluorescent detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels and two-dimensional (2-D) gels. Additionally, the technology is appropriate for the determination of protein kinase and phosphatase substrate preference. Other macromolecules, such as DNA, RNA, and sulfated glycans, fail to be detected with Pro-Q Diamond dye. The staining procedure is rapid, simple to perform, readily reversible and fully compatible with modern microchemical analysis procedures, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Pro-Q Diamond dye technology can detect as little as 1-2 ng of beta-casein, a pentaphosphorylated protein, and 8 ng of pepsin, a monophosphorylated protein. Fluorescence signal intensity correlates with the number of phosphorylated residues on the protein. Through combination of Pro-Q Diamond phosphoprotein stain with SYPRO(R) Ruby protein gel stain, Multiplexed Proteomics technology permits quantitative, dichromatic fluorescence detection of proteins in 2-D gels. This evolving discovery platform allows the parallel determination of protein expression level changes and altered post-translational modification patterns within a single 2-D gel experiment. The linear responses of the fluorescence dyes utilized, allow rigorous quantitation of changes over an unprecedented 500-1000-fold concentration range.

Proteomics of spermatogenesis: from protein lists to understanding the regulation of male fertility and infertility

“…To verify these results, we next used Pro-Q Diamond phosphostain (Invitrogen, Carlsbad, CA, USA) as a unique fluorescence-based detection system for the specific and sensitive analysis of protein and peptide phosphorylation status. 24 A total of 68 spots representing 52 proteins were stained, thereby confirming the presence of phosphorylated forms of these proteins in human testis. 22 Novel proteomics techniques are making it possible to identify large numbers of proteins in complex tissues.…”

Integrative proteomic analysis of the nucleus accumbens in rhesus monkeys following cocaine self-administration

“…[43][44][45][46][47] The specificity of Pro-Q Diamond was re-validated in the current study by examining the differential staining of known phosphorylated and nonphosphorylated proteins. Standard proteins and NAc cytosolic proteins from control and cocaine-treated monkeys were electrophoresed and the gels stained with Pro-Q Diamond and Sypro-Ruby.…”

State-of-the-art in phosphoproteomics

“…Commercially available phospho-stains are less sensitive than radioactive methods but the handling of these "inactive" reagents is far more convenient. These stains are mostly specific for certain types of phosphorylation such as all O-or just Ser/Thr-phosphorylations depending on the respective staining principle [12,13]. Furthermore, good compatibility to other staining methods and to MS is reported as no blotting is needed.…”