Bottom-up proteomics techniques provide a broad survey of the proteome with good precision and accuracy for quantitative comparisons between biological conditions. Briefly, proteins are extracted from the specimen, digested into peptides using a protease (most commonly trypsin), and analyzed by liquid chromatography coupled to mass spectrometry (LC-MS). At the Environmental Molecular Sciences Laboratory (EMSL), we use three main approaches for quantitative analyses: label-free, isobaric labeling, and targeted. When coupled with suitable standards, enrichment, or informatic approaches, all three approaches can be used for studying post-translational modifications (PTMs).
EMSL has developed cutting-edge capabilities for proteomics analysis of minute samples, including Nanodroplet Processing in One Pot for Trace Samples (nanoPOTS). These developments make it possible to obtain in-depth proteomics from samples containing < 1000 cells and, in some cases, down to the single-cell level. This approach can be coupled with cell sorting technologies to target specific cell populations or with laser capture microdissection to obtain spatially resolved proteome measurements.
- Supporting the Biomolecular Pathways Integrated Research Platform, these resources aid in the discovery of principles underlying the translation of genomic information into the metabolic pathways and regulatory networks that determine cellular function.
- Supporting the Biogeochemical Pathways Integrated Research Platform, these resources help to determine how microbes in soils react to changes in the soil environment and interact on the surrounding materials to play a role in nutrient cycling.
- Supporting the Rhizosphere Function Integrated Research Platform, these resources aid in the understanding of the molecular mechanisms involved in the determination of phenotypic plasticity in complex multifactorial ecosystems.
- Supporting the Cell Signaling and Communications Integrated Research Platform, these resources help determine how regulatory adjustments in one or a group of cells cascade through populations and communities of cells in a series of responses.
The EMSL User Program has a fleet of LC-MS instrumentation for bottom-up proteomics analysis.
For global and PTM analysis, we employ the following Thermo Scientific Orbitrap instruments:
- Fusion Lumos Tribrid (3)
- Eclipse Tribrid (1)
- Q Exactive Plus (4)
- Q Exactive HF-X (3).
For targeted proteomics analysis, we use the following triple quadrupole mass spectrometers:
- TSQ Altis (3)
- TSQ Vantage (4)
- Agilent G6490 (1).
Tips for success
- EMSL has full sample processing capability for bottom-up proteomics analysis. We advise that all sample processing steps, starting from protein extraction, are carried out at EMSL.
- Samples should be shipped on dry ice for preservation, and EMSL staff should be notified in advance to arrange for receiving.
- The use of detergents and other potential contaminants in sample handling should be discussed with EMSL staff.
- Study design and objectives should be discussed with your EMSL science point of contact to ensure the best approach is employed.
- EMSL employs three main approaches for bottom-up proteomics analysis. The advantages and requirements of each approach are briefly summarized in this table. These considerations should be considered when designing a bottom-up proteomics study.