Critical Comparison of Multidimensional Separation Methods for Increasing Protein Expression Coverage

We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC–MS/MS time was kept constant and the experiments were run in triplicate. We find that the most critical parameters are the data accumulation time, which defines the level of under-sampling and the avoidance of peptides from high expression level proteins eluting over the entire gradient

Quantitative Label-Free Phosphoproteomics of Six Different Life Stages of the Late Blight Pathogen Phytophthora infestans Reveals Abundant Phosphorylation of Members of the CRN Effector Family

The oomycete Phytophthora infestans is the causal agent of late blight in potato and tomato. Since the underlying processes that govern pathogenicity and development in P. infestans are largely unknown, we have performed a large-scale phosphoproteomics study of six different P. infestans life stages. We have obtained quantitative data for 2922 phosphopeptides and compared their abundance. Life-stage-specific phosphopeptides include ATP-binding cassette transporters and a kinase that only occurs in appressoria. In an extended data set, we identified 2179 phosphorylation sites and deduced 22 phosphomotifs. Several of the phosphomotifs matched consensus sequences of kinases that occur in P. infestans but not Arabidopsis. In addition, we detected tyrosine phosphopeptides that are potential targets of kinases resembling mammalian tyrosine kinases. Among the phosphorylated proteins are members of the RXLR and Crinkler effector families. The latter are phosphorylated in several life stages and at multiple positions, in sites that are conserved between different members of the Crinkler family. This indicates that proteins in the Crinkler family have functions beyond their putative role as (necrosis-inducing) effectors. This phosphoproteomics data will be instrumental for studies on oomycetes and host–oomycete interactions. The data sets have been deposited to ProteomeXchange (identifier PXD000433)

Relation of baseline triglycerides specie level to future adverse cardiovascular outcome adjusting for Framingham risk factors.

<p>Values are odds ratios (95% confidence intervals) for cardiovascular disease from multivariate adjusted binary logistic regressions performed with the Z score of a given triacylglyceride specie obtained after log transformation. BMI, body mass index; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; SBP, systolic blood pressure; TAG, triacylglyceride.</p

Baseline characteristics of the study samples.

<p>Values are mean±s.d. or percentage. <i>P</i> values were calculated using a t test for continuous variables and Pearson Chi-Square for binary variables.</p

LPC16∶0 and LPC20∶4 negatively correlate with CVD risk factors whereas SM38∶2 positively correlates with CVD risk factors.

<p>Partial correlations were performed between LPC16∶0, LPC20∶4, or SM38∶2 after log transformation and current known laboratory predictors for cardiovascular disease, adjusting for age and sex. BMI, body mass index; HbA1c, haemoglobin A1c; HDL, high-density lipoprotein cholesterol; Imtcca0, intima-media thickness of the common carotid artery at baseline; LDL, low-density lipoprotein cholesterol; LPC, lysophosphatidylcholine; SBP, systolic blood pressure; SM, sphingomyelin.</p

Association between the lipid profile and the risk allele of 8 CAD-associated gene variants.

<p>Heat map of regression coefficients obtained from linear regressions performed between the CAD-associated locus (with the CAD-associated allele coded) and the lipid species after log transformation adjusting for age and sex. *<i>P</i><0.05, ^o<i>P</i><0.01, ⁺<i>P</i><0.001.</p

Quantification by top-down lipidomics correlates with clinical parameters.

<p>Linear regression analysis of A) the total triglyceride content or B) the total cholesterol content determined by MS versus the value obtained by traditional clinical chemistry analysis. The total triglyceride content measured by MS is obtained by summing the abundances of all the individual TAG species and the total cholesterol content by summing the abundances of free cholesterol and all cholesteryl esters.</p