The study of protein phosphorylation is often referred to as phosphoproteomics, and it's one of the most studied and important posttranslational modification (PTM) in proteins. It occurs on serine, threonine, and tyrosine residues. This PTM plays a central role in regulating many cellular processes, including cell cycle progression, growth, and apoptosis. It also participates in innumerable signal transduction pathways. Because of the massive influence that phosphorylation has on biological processes, a huge emphasis has been placed on understanding its role in human health and disease.
Phosphopeptide enrichment reduces sample complexity and is required prior to MS due to the low stoichiometry and poor ionization of phosphopeptides. The Thermo Scientific High-Select Fe-NTA Phosphopeptide Enrichment Kit and High-Select TiO2 Phosphopeptide Enrichment Kit enable fast, selective enrichment of phosphorylated peptides for MS. The High-Select Fe-NTA Phosphopeptide Enrichment Kit uses iron-chelate resin spin columns, while the High-Select TiO2 Phosphopeptide Enrichment Kit uses TiO2 spin tips with optimized buffers. Both column types enrich 0.5-5 or 3 mg of total protein digest.
Typical phosphoproteomics workflows involve sample enrichment followed by mass spectrometry (MS) analysis using complementary fragmentation techniques (CID, HCD, EThcD, and ETD).These phosphoproteomics mass spectrometry workflows enable sensitive and conclusive structural elucidation of phosphorylation sites.
Reversed-phase liquid chromatography (RP-LC) enables successful separation of phosphopeptides ranging from a single protein to large-scale analysis. Thermo Scientific low flow RP-LC systems integrate seamlessly with available phosphoproteomics workflows and Orbitrap mass spectrometers. The Thermo Scientific EASY-nLC 1200 System and UltiMate 3000 RSLCnano System are the LC systems of choice for phosphoproteomics. The EASY-nLC 1200 ensures operational simplicity and high performance, while the UltiMate 3000 RSLCnano offers versatility and precision.
Today’s cutting-edge research pushes LC-MS to its limits. Obtaining high confidence insights to enable accurate resolution of subtle differences and avoid costly dead-ends is needed faster than ever before. The Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer surpasses these limits with new innovations that deliver the ultimate flexibility to expand experimental scope, and with built-in intelligence to ensure the highest data quality and confidence. One system provides maximum insights, so you productively go beyond today’s discovery.
Data-dependent decision tree (DDDT) logic is available on all Thermo Scientific hybrid linear ion trap-Orbitrap mass spectrometers, including the Thermo Scientific Orbitrap Fusion Lumos Mass Spectrometer. The DDDT method improves phosphopeptide identifications and increases throughput when compared to analyses using only CID and ETD. Newly introduced EThcD enables more thorough fragmentation of unmodified and phosphorylated peptides than HCD or ETD alone, increasing confidence in phosphorylation site localization.
Phosphorylation occurs on serine, threonine, and tyrosine residues, posing a challenge for accurate phosphopeptide data analysis. The solution is high-resolution accurate-mass (HRAM) MS, which not only generates accurate data but also reduces false discovery rates. Thermo Scientific Proteome Discoverer Software has all the necessary tools for data mining of HRAM MS mixed raw files, including those containing multiple fragmentation spectra. The software's novel feature, IMP-ptmRS, offers a phosphorylation site confidence measurement algorithm that increases phosphoproteome coverage from LC-MS/MS data sets.
Proteins for phosphoproteomic analysis come from a variety of sources and must first be extracted and denatured, then reduced, alkylated and digested into peptides. Phosphopeptides are then enriched before they can be analyzed by LC-MS/MS on Orbitrap-based mass spectrometers. The phosphopeptides are identified, and sites of phosphorylation are assigned using Proteome Discoverer software with IMP-ptmRS.
Read how deep phosphoproteome sequencing was enabled with a novel enrichment approach, how utilization of a fractionation kit reduced overall complexity for greater coverage of the phosphoproteome, and how combining the techniques with a TMT workflow resulted in quantification of nearly 24,000 phosphopeptides in eleven different samples.
High-Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) can be used to optimize ion selectivity and reduce chemical backgrounds during mass spectrometry assays. FAIMS successfully prevents interferences from co-eluting compounds and increases signal to noise ratios by 100-fold or higher.