Determining the whole from the sum of its parts
Qualitative or bottom-up proteomics is still the mainstay for most proteomics experiments. The objective of this workflow is to identify as many protein components in a biological sample as possible. The approach taken in bottom-up proteomics is to first digest the protein to its peptide components, then separate those components using liquid chromatography (LC), and finally identify the peptides by MS. The resulting sequence data are used to determine the original components of the sample.
The "workhorse" of proteomics is bottom-up analysis, and the majority of proteomics workflows involve the use of a bottom-up strategy. In this approach, proteins are first subjected to enzymatic digestion typically using trypsin. This generates peptides, which are then separated using one or more LC technologies. The LC eluent is charged via electrospray ionization, after which the peptides are fragmented within the mass spectrometer and identified.
Deriving high quality peptide identification from complex biological samples over a wide dynamic range is a challenging but necessary aspect of bottom-up proteomics. Fortunately, high resolution accurate mass (HRAM) Orbitrap mass spectrometers offer advanced mass resolution and accuracy, as well as fragmentation and speed. Multiple fragmentation techniques (CID, HCD, ETD, and EThcD) ensure maximal sequence coverage of analyzed peptides. These combined Orbitrap system qualities enable higher and more detailed bottom-up protein identifications from complex proteomics samples than ever before.
Once the raw analysis files are generated, Thermo Scientific proteomics software mines the rich HRAM Orbitrap MS data and converts large data sets into meaningful insights.
The Thermo Scientific Orbitrap Fusion Lumos Tribrid Mass Spectrometer possesses the highest resolving power of all Orbitrap-based instruments (up to 500,000 @ m/z 200), achieving high detection limits and selectivity in full-scan MS mode, which permits quick and thorough analysis of complex matrices. The system's brighter ion source and Advanced Quadrupole Technology enable low limits of detection, while its integrated Universal Method, powered by ADAPT technology, generates high-quality results from unknown concentration samples.
The Thermo Scientific Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer combines an HRAM ultra-high-field Orbitrap analyzer with high performance quadrupole precursor ion selection to deliver unsurpassed speed while maintaining sensitivity. Compared to previous Q Exactive generations (Q Exactive MS and Q Exactive Plus MS), the Q Exactive HF MS realizes a 1.8x higher resolution power at the same transient length and a 2x higher scan speed at almost the same resolution setting (15,000 vs 17,500 @ m/z 200). This shortens analysis time two-fold, yet delivers almost the same number of unique peptide identifications.
Thermo Scientific ProteinCenter Software facilitates proteomics data interpretation by quickly extracting meaningful biological information from multiple and complex data sets. Protein and peptide data sets can be compared from different times and experiments, and redundant data can be excluded. Over 10 million non-redundant proteins and over 20 public databases are included in the ProteinCenter software database.
Thermo Scientific Proteome Discoverer Software is the most comprehensive data analysis platform for bottom-up proteomics research. The software features a comprehensive set of tools for data mining of CID, HCD, ETD, and EThcD fragmentation spectra using the SEQUEST, Mascot, and Byonic search engines, including spectra obtained from mixed raw files using multiple fragmentation modes in a single run. As a result, Proteome Discoverer can deliver confident PTM identification and site localization information.