44 research outputs found
Peptide retention time prediction for immobilized artificial membrane phosphatidylcholine stationary phase: method development and preliminary observations
Development of the first peptide retention prediction model for immobilized artificial membrane phosphatidylcholine (IAM.PC) stationary phase is reported. 2D liquid chromatography coupled to tandem mass spectrometry (2D LC-MS/MS) analysis of a whole cell lysate of S. cerevisiae yielded a retention dataset of ~29,500 tryptic peptides; sufficient for confident assignment of retention coefficients which determine the contribution of individual amino acids in peptide retention. Retention data from the first dimension was used for the modelling: an IAM.PC.DD2 column, with pH 7.4 ammonium bicarbonate, and a water/acetonitrile gradient. Peptide separation using the IAM.PC.DD2 phase was compared to a standard C18 phase (Luna C18(2)). There was a significant reduction in peptide retention (~14 % acetonitrile on average), indicating that the phosphatidylcholine stationary phase is significantly more hydrophilic. In comparison to the C18 phase, a substantial increase was found in the relative retention contribution for the positively charged Arg and Lys, and the aromatic Tyr, Trp and His residues. A decrease in retention contribution was observed for the negatively charged Asp and Glu. This indicates an involvement of electrostatic interactions with the glycerophosphate functional groups, and possibly, delocalization effects from hydrogen bonds between the phosphate group and the aromatic side chains in the separation mechanism
Global changes in the proteome of Cupriavidus necator H16 during poly-(3-hydroxybutyrate) synthesis from various biodiesel by-product substrates
Additional file 1: Table S1. P-scores of proteomic runs of C. necator H16 grown with different substrates
Retention Order Reversal of Phosphorylated and Unphosphorylated Peptides in Reversed-Phase LC/MS
Protein phosphorylation is one of the most ubiquitous post-translational modifications in humans, and trypsin-digested phosphorylated peptides have been analyzed by reversed phase LC/MS using C18-silica columns under acidic conditions to profile human phosphoproteomes. Here, we report that phosphopeptides generally exhibit stronger retention than their unphosphorylated counterparts when C18-silica columns are used with acetic acid or formic acid as an ion-pairing reagent, whereas the retention order is reversed when less hydrophobic stationary phases such as C4-silica columns are employed. Similarly the retention reversal is observed when more hydrophobic ion-pairing reagents such as trifluoroacetic acid are used with C18-silica columns. These phenomena could be explained by the smaller S-values of phosphopeptides in linear solvation strength theory, based on the reduced net charge caused by intramolecular interaction between phosphate and basic groups
Peptide Retention Time Prediction in Hydrophilic Interaction Liquid Chromatography: Data Collection Methods and Features of Additive and Sequence-Specific Models
The
development of a peptide retention prediction model for hydrophilic
interaction liquid chromatography (XBridge Amide column) is described
for a collection of ∼40 000 tryptic peptides. Off-line
2D LC-MS/MS analysis (HILIC-RPLC) of <i>S. cerevisiae</i> whole cell lysate has been used to acquire retention information
for a HILIC separation. The large size of the optimization data set
(more than 2 orders of magnitude compared to previous reports) permits
the accurate assignment of hydrophilic retention coefficients of individual
amino acids, establishing both the effects of amino acid position
relative to peptide termini and the influence of peptide secondary
structure in HILIC. The accuracy of a simple additive model with peptide
length correction (<i>R</i><sup>2</sup> value of ∼0.96)
was found to be much higher compared to an algorithm of similar complexity
applied to RPLC (∼0.91). This indicates significantly smaller
influence of peptide secondary structure and interactions with counterions
in HILIC. Nevertheless, sequence-specific features were found. Helical
peptides that tend to retain stronger than predicted in RPLC exhibit
negative prediction errors using an additive HILIC model. N-cap helix
stabilizing motifs, which increase retention of amphipathic helical
peptides in RP, reduce peptide retention in HILIC independently of
peptide amphipathicity. Peptides carrying multiple Pro and Gly (residues
with lowest helical propensity) retain stronger than predicted. We
conclude that involvement of the peptide backbone’s carbonyl
and amide groups in hydrogen-bond stabilization of helical structures
is a major factor, which determines sequence-dependent behavior of
peptides in HILIC. The incorporation of observed sequence dependent
features into our Sequence-Specific Retention Calculator HILIC model
resulted in 0.98 <i>R</i><sup>2</sup> value correlation,
significantly exceeding the predictive performance of all RP and HILIC
models developed for complex mixtures of tryptic peptides
In situ activity-based protein profiling of serine hydrolases in E. coli
A fluorophosphonate based alkyne activity probe was used for the selective labeling of active serine hydrolases in intact Escherichia coli cells. A biotin-azide tag was subsequently attached to the alkyne functionality of the probe with copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Comparison of proteins from in-cell and lysate labeled preparations suggested qualitatively similar patterns of reactivity in both preparations. Approximately 68%, 30 of the total 44 serine hydrolases detectable in E. coli were labeled with the probe indicating significant coverage with a single probe. The methods described here offer a useful tool for profiling and monitoring serine hydrolase activity in situ
Proteomic Shifts Reflecting Oxidative Stress and Reduced Capacity for Protein Synthesis, and Alterations to Mitochondrial Membranes in Neurospora crassa Lacking VDAC
Voltage-dependent anion-selective channels (VDAC) maintain the bidirectional flow of small metabolites across the mitochondrial outer membrane and participate in the regulation of multiple cellular processes. To understand the roles of VDAC in cellular homeostasis, preliminary proteomic analyses of S100 cytosolic and mitochondria-enriched fractions from a VDAC-less Neurospora crassa strain (ΔPor-1) were performed. In the variant cells, less abundant proteins include subunits of translation initiation factor eIF-2, enzymes in the shikimate pathway leading to precursors of aromatic amino acids, and enzymes involved in sulfate assimilation and in the synthesis of methionine, cysteine, alanine, serine, and threonine. In contrast, some of the more abundant proteins are involved in electron flow, such as the α subunit of the electron transfer flavoprotein and lactate dehydrogenase, which is involved in one pathway leading to pyruvate synthesis. Increased levels of catalase and catalase activity support predicted increased levels of oxidative stress in ΔPor-1 cells, and higher levels of protein disulfide isomerase suggest activation of the unfolded protein response in the endoplasmic reticulum. ΔPor-1 cells are cold-sensitive, which led us to investigate the impact of the absence of VDAC on several mitochondrial membrane characteristics. Mitochondrial membranes in ΔPor-1 are more fluid than those of wild-type cells, the ratio of C18:1 to C18:3n3 acyl chains is reduced, and ergosterol levels are lower. In summary, these initial results indicate that VDAC-less N. crassa cells are characterized by a lower abundance of proteins involved in amino acid and protein synthesis and by increases in some associated with pyruvate metabolism and stress responses. Membrane lipids and hyphal morphology are also impacted by the absence of VDAC