A Simple Protocol To Routinely Assess the Uniformity of Proteomics Analyses

Mass-spectrometry-based proteomic approaches are increasingly applied to biological and clinical studies. Initially used by specialized laboratories, the technology has matured and gained acceptance by the community, using various analytical processes and platforms. To facilitate data comparison and integration across laboratories, there is a need to harmonize analytical processes to ensure the generation of reliable proteomic data sets. This is especially critical in the context of large initiatives, such as the <i>Human Proteome Project</i> promoted by the Human Proteome Organization (HUPO). Quality control is a first step toward the harmonization of proteomics data sets. We have developed a procedure to routinely assess the uniformity of proteomics analyses. It relies on a simple protocol based on three proteins and two sets of isotopically labeled peptides, one being added prior to tryptic digestion and the second one prior to liquid chromatography–mass spectrometry (LC–MS) analysis. The proposed method evaluates in a single step both the sample preparation, by measuring the relative amounts of endogenous peptides and their isotopically labeled counterparts, and the LC–MS platform performance, by monitoring the main LC–MS attributes for reference peptides. The procedure is simple and easy to implement into routine workflows typically employed by the proteomics community

Phosphoproteome Exploration Reveals a Reformatting of Cellular Processes in Response to Low Sterol Biosynthetic Capacity in <i>Arabidopsis</i>

Sterols are membrane-bound isoprenoid lipids that are required for cell viability and growth. In plants, it is generally assumed that 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) is a key element of their biosynthesis, but the molecular regulation of that pathway is largely unknown. In an attempt to identify regulators of the biosynthetic flux from acyl-CoA toward phytosterols, we compared the membrane phosphoproteome of wild-type <i>Arabidopsis thaliana</i> and of a mutant being deficient in HMGR1. We performed a N-terminal labeling of microsomal peptides with a trimethoxyphenyl phosphonium (TMPP) derivative, followed by a quantitative assessment of phosphopeptides with a spectral counting method. TMPP derivatization of peptides resulted in an improved LC–MS/MS detection due to increased hydrophobicity in chromatography and ionization efficiency in electrospray. The phosphoproteome coverage was 40% higher with this methodology. We further found that 31 proteins were in a different phosphorylation state in the <i>hmgr1–1</i> mutant as compared with the wild-type. One-third of these proteins were identified based on novel phosphopeptides. This approach revealed that phosphorylation changes in the <i>Arabidopsis</i> membrane proteome targets major cellular processes such as transports, calcium homeostasis, photomorphogenesis, and carbohydrate synthesis. A reformatting of these processes appears to be a response of a genetically reduced sterol biosynthesis