Methods in molecular biology
-
Protein post-translational modifications (PTMs) are crucial for signal transduction in cells. In order to understand key cell signaling events, identification of functionally important PTMs, which are more likely to be evolutionarily conserved, is necessary. In recent times, high-throughput mass spectrometry (MS) has made quantitative datasets in diverse species readily available, which has led to a growing need for tools to facilitate cross-species comparison of PTM data. ⋯ Here, we describe an automated web-based tool, PhosphOrtholog, that accurately maps annotated and novel orthologous PTM sites from high-throughput MS-based experimental data obtained from different species without relying on existing PTM databases. Identification of conserved PTMs across species from large-scale experimental data increases our knowledgebase of evolutionarily conserved and functional PTM sites that influence most biological processes. In this Chapter, we illustrate with examples how to use PhosphOrtholog to map novel PTM sites from cross-species MS-based phosphoproteomics data.
-
Multiplex assays that allow the simultaneous measurement of multiple analytes in small sample quantities have developed into a widely used technology. Their implementation spans across multiple assay systems and can provide readouts of similar quality as the respective single-plex measures, albeit at far higher throughput. Multiplex assay systems are therefore an important element for biomarker discovery and development strategies but analysis of the derived data can face substantial challenges that may limit the possibility of identifying meaningful biological markers. This chapter gives an overview of opportunities and challenges of multiplexed biomarker analysis, in particular from the perspective of machine learning aimed at identification of predictive biological signatures.
-
High resolution mass spectrometry has revolutionized proteomics over the past decade, resulting in tremendous amounts of data in the form of mass spectra, being generated in a relatively short span of time. The mining of this spectral data for analysis and interpretation though has lagged behind such that potentially valuable data is being overlooked because it does not fit into the mold of traditional database searching methodologies. ⋯ In this chapter, we propose a methodology to integrate de novo peptide sequencing using three commonly available software solutions in tandem, complemented by homology searching, and manual validation of spectra. This simplified method would allow greater use of de novo sequencing approaches and potentially greatly increase proteome coverage leading to the unearthing of valuable insights into protein biology, especially of organisms whose genomes have been recently sequenced or are poorly annotated.
-
The protocol herein describes a robust and proven method for the measurement of pseudokinase-ligand interaction using a fluorescence-based thermal shift assay (TSA). Pseudokinases are kinase-like proteins that have recently emerged as crucial regulatory modules of signal transduction pathways and may well represent a novel class of drug targets. However, unlike kinases, the regulatory activity of pseudokinases is mainly conferred through protein-protein interactions. ⋯ Ligand binding to a protein is known to increase its thermal stability, which is reflected by a shift between the thermal denaturation curves of the unliganded protein and the liganded protein. Here, we illustrate the utility of the method with the pseudokinases, ErbB3/HER3, ILK, ROP5Bi, JAK1, JAK2, TYK2, MLKL, STRAD, TRIB1, VRK3, and ROR1. This method can also be used to determine optimal buffer conditions that may increase protein stability and can be tailored to other protein families.
-
Direct oral anticoagulants (DOACs) can be quantified using methods that can be performed in any clinical or research laboratory using manual or automated instrument platforms. Dabigatran etexilate, the oral direct thrombin inhibitor, can be quantified by drug-calibrated clot or chromogenic-based assays using either thrombin or ecarin as substrates. Oral direct anti-Xa inhibitors, such as rivaroxaban, apixaban, and edoxaban, can be quantified with drug-calibrated anti-Xa kits or reagents as typically used for measuring heparins (unfractionated, low molecular weight, or pentasaccharides).