Bottom-up Proteomics

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) Thermo Scientific 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.

Bottom-up proteomics instruments
 Orbitrap Eclipse Tribrid Mass Spectrometer
Orbitrap Eclipse Tribrid Mass Spectrometer
Orbitrap Exploris 480 Mass Spectrometer
Orbitrap Exploris 480 Mass Spectrometer
Q Exactive HF
Orbitrap Exploris 240 Mass Spectrometer
AdvantagesObtain maximum insights on your most complex molecules and biological systems, from whole proteome profiling and quantitation, structural characterization to multiplexed single-cell proteomics. With new innovations that deliver the ultimate flexibility in experimental scope, it accelerates your path to new, impactful results, so you can drive your science beyond today’s discovery.Obtain maximum quantitative insights from untargeted proteome profiles to targeted proteomics experiments with industry leading single-cell sensitivity and extraordinary accuracy, precision and simplicity. With curated workflows that deliver greater usability, it accelerates your path to large-scale studies, delivering proven high data quality and time savings, so you can go beyond faster to actionable outcomes.Expand your capabilities from small- to large-scale studies across a variety of applications from protein identification, quantitation, to multiplexing proteomics studies. With optimized methods, it delivers a fast turnaround of sample to results with operational simplicity. Best-in-class performance, all within a compact footprint, so you can go beyond with everyday versatility.
Resolving Power7,500-500,000 FWHM at m/z 200 (1M option)480,000 at m/z 195Up to 240,000 FWHM at m/z 200
Scan SpeedUp to 40Hz
Mass Range50 to 6,000 (Up to 8,000 with HMRn)40 to 6,000 m/z (up to 8,000 m/z with the BioPharma option)
Dynamic Range>5000 with a single OTMS spectrum>5000:1
Mass AccuracyInternal <1 ppm RMS; External: <3 ppm RMS

Software for bottom-up proteomics

Utilize powerful data mining tools

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 HT, 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 software can deliver confident PTM identification and site localization information.

Bottom-up proteomics workflow

Style Sheet for Global Design System
CMD SchemaApp code