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.
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Learn about the bottom-up proteomics method
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.
Orbitrap Eclipse Tribrid Mass Spectrometer
Orbitrap Fusion Lumos Tribrid Mass Spectrometer
Orbitrap Exploris 480 Mass Spectrometer
Q Exactive HF hybrid Quadrupole-Orbitrap Mass Spectrometer
|Advantages||Go beyond today's discovery with new innovations that deliver the ultimate flexibility in one system, plus pre-defined data acquisition methods and built-in intelligence for workflows, including TMT, for the highest quality MS data.||Experience the highest possible resolution, achieving high detection limits and selectivity in full-scan MS mode, which permits quick and thorough analysis of complex matrices.||Making genius simpler with a compact footprint, empowerment for all users and a data-aware quantitation scan mode for targeted proteome analysis in real time, filling the gap for large-scale target profiling, reducing hassles and increasing productivity.||Realize unsurpassed resolution and speed by combining an HRAM ultra-high-field Orbitrap analyzer with high performance quadrupole precursor ion selection to deliver unsurpassed speed while maintaining sensitivity.|
|Resolving Power||7,500-500,000 FWHM at m/z 200 (1M option)||480,000 at m/z 195||Up to 240,000 FWHM at m/z 200|
|Scan Speed||Up to 40Hz|
|Mass Range||50 to 6,000 (Up to 8,000 with HMRn)||50 to 6,000||40 to 6,000 m/z (up to 8,000 m/z with the BioPharma option)||50 to 6,000 m/z (up to 8,000 with BioPharma Option)|
|Dynamic Range||>5000 with a single OTMS spectrum||>5000:1|
|Mass Accuracy||Internal <1 ppm RMS; External: <3 ppm RMS|
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.
Software for bottom-up proteomics
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.