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

Data_Sheet_1_In Vitro Evolution of Antibodies Inspired by In Vivo Evolution.PDF

<p>In vitro generation of antibodies often requires variable domain sequence evolution to adapt the protein in terms of affinity, specificity, or developability. Such antibodies, including those that are of interest for clinical development, may have their origins in a diversity of immunoglobulin germline genes. Others and we have previously shown that antibodies of different origins tend to evolve along different, preferred trajectories. Apart from substitutions within the complementary determining regions, evolution may also, in a germline gene-origin-defined manner, be focused to residues in the framework regions, and even to residues within the protein core, in many instances at a substantial distance from the antibody’s antigen-binding site. Examples of such germline origin-defined patterns of evolution are described. We propose that germline gene-preferred substitution patterns offer attractive alternatives that should be considered in efforts to evolve antibodies intended for therapeutic use with respect to appropriate affinity, specificity, and product developability. We also hypothesize that such germline gene-origin-defined in vitro evolution hold potential to result in products with limited immunogenicity, as similarly evolved antibodies will be parts of conventional, in vivo-generated antibody responses and thus are likely to have been seen by the immune system in the past.</p

Multimodel Pathway Enrichment Methods for Functional Evaluation of Expression Regulation

Functional analysis of quantitative expression data is becoming common practice within the proteomics and transcriptomics fields; however, a gold standard for this type of analysis has yet not emerged. To grasp the systemic changes in biological systems, efficient and robust methods are needed for data analysis following expression regulation experiments. We discuss several conceptual and practical challenges potentially hindering the emergence of such methods and present a novel method, called FEvER, that utilizes two enrichment models in parallel. We also present analysis of three disparate differential expression data sets using our method and compare our results to other established methods. With many useful features such as pathway hierarchy overview, we believe the FEvER method and its software implementation will provide a useful tool for peers in the field of proteomics. Furthermore, we show that the method is also applicable to other types of expression data

Automated Selected Reaction Monitoring Software for Accurate Label-Free Protein Quantification

Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of <i>Streptococcus pyogenes</i> we achieved a technical variability across the entire proteome abundance range of 6.5–19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology

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)