Technology
Trapped Ion Mobility Mass Spectrometry (TIMS) Drives High-throughput Phosphoproteomics Research Unlike genomic and transcriptomic research, the measurement technologies for proteomics are still evolving, and the complete analysis of a proteome has not yet been achieved. The end goal of proteomics studies is not just to identify all the proteins that can be expressed, but also to uncover cellular protein events such as protein expression/ degradation, protein localisation, protein interaction, protein posttranslational modifications (PTM), and protein processing/splicing. Protein phosphorylation is an abundant form of reversible PTM and plays a vital role in various cellular processes, such as protein synthesis, cell division, signal transduction, cell growth, development, and ageing1. As such, it regulates important metabolic, hormonal, developmental, and stress responses. Atypical phosphorylation can contribute to a range of disease states, including cancer and diabetes. Phosphoproteomics – the characterisation of proteins with phosphorylated PTMs – is therefore an important tool for obtaining insights into health and disease. In cellular signal transduction networks, reversible phosphorylation is one of the key events in transducing a signal into the nucleus to control gene expression. Approximately 30% of human proteins were previously estimated to be phosphorylated, but researchers at the Laboratory of Molecular and Cellular BioAnalysis at Kyoto University developed a highly selective enrichment method for phosphopeptides and, when applied to proteome-wide acquisition of cellular phosphorylation status, revealed that at least 70% of human proteins are phosphorylated2,3. Recent technological advances have enabled the use of mass spectrometry (MS) in phosphoproteomic approaches to address the scope of phosphorylation. We have developed phosphoproteomics methods to carry out in vivo phosphoproteome profiling of kinase-targeting drugs, which would facilitate drug discovery 68 INTERNATIONAL PHARMACEUTICAL INDUSTRY
and development for cancer therapy, as well as enable the exploration of the functional analysis of newly discovered phosphorylated molecules. MS-based Phosphoproteomics Deep characterisation and quantitative analysis of proteins and PTMs are critical to understanding signalling pathways and abnormal disease states. Developments in high-resolution mass spectrometers and specific enrichment of phosphorylated peptides tailored for the global analysis of protein phosphorylation represent powerful tools for molecular and cellular biologists studying signal transduction pathways. Despite these advances, identifying PTMs remains significantly more challenging compared with unmodified peptides, as they often occur at low abundances and the differences in protein phosphorylation span several orders of magnitude, driving the need for instruments with higher sensitivity and increased peak capacities. Protein expression varies depending on the genetic background of an individual, but also on time, localisation, and as a physiological response to external stimuli (stress, disease, ageing, effort, etc.). Moreover, because of the combined effects of alternative splicing, point mutation, PTM and endogenous proteolysis, a given protein (gene expression product) can be expressed as many different proteoforms, each having a dedicated biological activity. The sophistication of modern instrumentation enables the identification of tens of thousands of phosphopeptides in a singleshot liquid chromatography coupled with mass spectrometry (LC-MS) run, but the percentage occurring as positional isomers is unknown. The combination of ion mobility spectrometry (IMS) with MS is a wellestablished technique that has shown considerable potential for improving peptide identification, providing structural information that is complementary to LC and MS. IMS-MS separates ions based on differences in their shape (IMS) and mass (MS), delivering information on the threedimensional (3D) structure of an ion. This added ability to separate ions by differences
in conformation makes it possible to separate isobaric and isomeric species, such as phosphopeptide positional isomers (i.e., peptides that differ only by the residue that is phosphorylated), which are not easily distinguished by MS techniques alone4. The commercialisation of trapped ion mobility spectrometry (TIMS) in 2016 built on the advances in IMS technology made over previous years. TIMS enables the interrogation and manipulation of mobility separated ion populations in the gas-phase, with high efficiency, duty cycle and high resolving power in millisecond-second timescales, and with the possibility to measure collisional cross section (CCS) using first principles that can be further utilised for structural assignments5, in CCS-aware workflows. The addition of TIMS provides a fourth dimension that is complementary to the previously used mass, intensity and retention time dimensions, resulting in 4D-Protoemics methods (Figure 1). TIMS is most often coupled with a time-of-flight (TOF) mass analyser to capitalise on its highspeed capabilities. A fractionation approach is often used to obtain deeper proteome coverage, but this method is time-consuming, and if many clinical samples are analysed, the timeframe is not realistic. Modern TIMS-QTOF MS systems utilising parallel accumulation – serial fragmentation (PASEF) can provide the necessary high speed and increased sensitivity. The novel design allows for ions to be accumulated in the front section, while ions in the rear section are sequentially released depending on their ion mobility. Modern TIMS-QTOF MS with PASEF can provide sequencing speed > 100 Hz without losing sensitivity or resolution by synchronising the quadrupole isolation mass window with the elution time of the specific peptide packages from the TIMS funnel. Our research found that PASEF can effectively increase MS/MS acquisition rates to reach new depths in phosphoproteomics, improving both identification and confidence in results. The high speed and sensitivity of the PASEF acquisition mode also facilitates high throughput on a TIMS-QTOF MS system. Autumn 2020 Volume 12 Issue 3
