RIBAR and xRIBAR: methods for reproducible relative MS/MS-based label-free protein quantification

Mass spectrometry-driven proteomics is increasingly relying on quantitative analyses for biological discoveries. As a result, different methods and algorithms have been developed to perform relative or absolute quantification based on mass spectrometry data. One of the most popular quantification methods are the so-called label-free approaches, which require no special sample processing, and can even be applied retroactively to existing data sets. Of these label-free methods, the MS/MS-based approaches are most often applied, mainly because of their inherent simplicity as compared to MS-based methods. The main application of these approaches is the determination of relative protein amounts between different samples, expressed as protein ratios. However, as we demonstrate here, there are some issues with the reproducibility across replicates of these protein ratio sets obtained from the various, MS/MS-based label-free methods, indicating that the existing methods are not optimally robust. We therefore present two new Methods (called RIBAR and xRIBAR) that use the available MS/MS data more effectively, achieving increased robustness. Both the accuracy and the precision of our novel methods are analyzed and compared to the existing methods to illustrate the increased robustness of our new methods over existing ones

Positional proteomics reveals differences in N-terminal proteoform stability

To understand the impact of alternative translation initiation on a proteome, we performed a proteome-wide study on protein turnover using positional proteomics and ribosome profiling to distinguish between N-terminal proteoforms of individual genes. By combining pulsed SILAC with N-terminal COFRADIC, we monitored the stability of 1,941 human N-terminal proteoforms, including 147N-terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine. N-terminally truncated proteoforms were less abundant than canonical proteoforms and often displayed altered stabilities, likely attributed to individual protein characteristics, including intrinsic disorder, but independent of N-terminal amino acid identity or truncation length. We discovered that the removal of initiator methionine by methionine aminopeptidases reduced the stability of processed proteoforms, while susceptibility for N-terminal acetylation did not seem to influence protein turnover rates. Taken together, our findings reveal differences in protein stability between N-terminal proteoforms and point to a role for alternative translation initiation and co-translational initiator methionine removal, next to alternative splicing, in the overall regulation of proteome homeostasis

Proteogenomics in aid of host-pathogen interaction studies : a bacterial perspective

By providing useful tools to study host-pathogen interactions, next-generation omics has recently enabled the study of gene expression changes in both pathogen and infected host simultaneously. However, since great discriminative power is required to study pathogen and host simultaneously throughout the infection process, the depth of quantitative gene expression profiling has proven to be unsatisfactory when focusing on bacterial pathogens, thus preferentially requiring specific strategies or the development of novel methodologies based on complementary omics approaches. In this review, we focus on the difficulties encountered when making use of proteogenomics approaches to study bacterial pathogenesis. In addition, we review different omics strategies (i.e., transcriptomics, proteomics and secretomics) and their applications for studying interactions of pathogens with their host

The iceLogo web server and SOAP service for determining protein consensus sequences

he iceLogo web server and SOAP service implement the previously published iceLogo algorithm. iceLogo builds on probability theory to visualize protein consensus sequences in a format resembling sequence logos. Peptide sequences are compared against a reference sequence set that can be tailored to the studied system and the used protocol. As such, not only over-but also underrepresented residues can be visualized in a statistically sound manner, which further allows the user to easily analyse and interpret conserved sequence patterns in proteins. The web application and SOAP service can be found free and open to all users without the need for a login on http://iomics.ugent.be/icelogoserver/main.html

N-terminal proteoforms in human disease

The collection of chemically different protein variants, or proteoforms, by far exceeds the number of protein-coding genes in the human genome. Major contributors are alternative splicing and protein modifications. In this review, we focus on those proteoforms that differ at their N termini with a molecular link to disease. We describe the main underlying mechanisms that give rise to such N-terminal proteoforms, these being splicing, initiation of protein translation, and protein modifications. Given their role in several human diseases, it is becoming increasingly clear that several of these N-terminal proteoforms may have potential as therapeutic interventions and/or for diagnosing and prognosing their associated disease

Bacterial genetic engineering by means of recombineering for reverse genetics

Serving a robust platform for reverse genetics enabling the in vivo study of gene functions primarily in enterobacteriaceae, recombineering -or recombination-mediated genetic engineering- represents a powerful and relative straightforward genetic engineering tool. Catalyzed by components of bacteriophage-encoded homologous recombination systems and only requiring short ~40 to 50 base homologies, the targeted and precise introduction of modifications (e.g., deletions, knockouts, insertions and point mutations) into the chromosome and other episomal replicons is empowered. Furthermore, by its ability to make use of both double- and single-stranded linear DNA editing substrates (e.g., PCR products or oligonucleotides, respectively), lengthy subcloning of specific DNA sequences is circumvented. Further, the more recent implementation of CRISPR-associated endonucleases has allowed for more efficient screening of successful recombinants by the selective purging of non-edited cells, as well as the creation of markerless and scarless mutants. In this review we discuss various recombineering strategies to promote different types of gene modifications, how they are best applied, and their possible pitfalls

Improved glucocorticoid receptor ligands : fantastic beasts, but how to find them?

Exogenous glucocorticoids are widely used in the clinic for the treatment of inflammatory disorders and hematological cancers. Unfortunately, their use is associated with debilitating side effects, including hyperglycemia, osteoporosis, mood swings, and weight gain. Despite the continued efforts of pharma as well as academia, the search for so-called selective glucocorticoid receptor modulators (SEGRMs), compounds with strong anti-inflammatory or anti-cancer properties but a reduced number or level of side effects, has had limited success so far. Although monoclonal antibody therapies have been successfully introduced for the treatment of certain disorders (such as anti-TNF for rheumatoid arthritis), glucocorticoids remain the first-in-line option for many other chronic diseases including asthma, multiple sclerosis, and multiple myeloma. This perspective offers our opinion on why a continued search for SEGRMs remains highly relevant in an era where small molecules are sometimes unrightfully considered old-fashioned. Besides a discussion on which bottlenecks and pitfalls might have been overlooked in the past, we elaborate on potential solutions and recent developments that may push future research in the right direction