Our Expertise

Protein and peptide identification

Protein samples are processed with enzymatic digestion and resulting peptides are analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The Platform offers protein identification from gel bands, affinity/proximity-enriched protein complexes to complex protein mixtures from cells, body fluids and tissues. For highly complex protein mixtures, peptide fractionation may be applied to reduce sample complexity and increase the proteome coverage.

High-throughput protein quantification

For most biological questions, information on the presence or absence of proteins in a given sample is not sufficient, and quantitative information of protein and/or Post-Translational Modification (PTM) abundance changes is required to compare between treatment conditions, strains, time points and/or disease states. Protein quantification can be analyzed by label-free and label-based strategies. Each strategy has its advantages and limits and the Platform designs the optimal workflow for each new project accordingly.

Label-free quantification with Data Independent Acquisition Mass Spectrometry (DIA-MS)

The Data Independent Acquisition Mass Spectrometry (DIA-MS) workflow acquires data in a discovery mode and analyzes them by targeted analysis using reference spectra. This method provides comprehensive measurement of all detectable proteins with high reproducibility across large sample cohorts. The DIA-MS method is ideal for highly multiplexed protein quantification and enables identification of differentially expressed proteins on a proteome level.

Label-based quantification

Label-based protein quantification can be performed in different manners, including metabolic labeling and chemical labeling, etc.

Stable isotope labelling by amino acids in cell culture (SILAC) depends on metabolic incorporation of isotope-containing amino acids (“light”, “medium” or “heavy”) and labels cellular proteomes through metabolic processes. The isotopically labeled amino acids are chemically identical, but the proteins/peptides are distinguishable by mass spectrometry. Thus, light, medium and heavy cell populations are mixed and analyzed by LC-MS/MS.  SILAC provides accurate protein quantification in any cell culture system. 

Alternatively, protein quantification can be performed using chemical labeling methods, such as Tandem Mass Tag (TMT) labeling. TMT labeling utilizes isobaric reagents to label the primary amines of peptides and enables identification and quantification of proteins in a multiplexed (6-plex to 11-plex) manner. Proteins from samples to be analyzed are digested independently, and resulting peptides are labeled with different TMT reagents and mixed with equal amounts. The combined peptide mixture is analyzed by LC-MS/MS for protein identification and quantification.

Identification and localization of PTMs

PTMs are known to be crucial in regulation of signal transduction and are involved in many diseases. The Platform offers identification of PTMs including common modifications, such as phosphorylation, acetylation, as well as potential modifications from specific interest molecules (Discussion with Platform members is suggested before experiment). A probability score is computed for every modified peptide and a localization score is provided for several modified amino acid positions. Combined with the quantification methods, abundance of proteins as well as PTMs could be assessed across samples.

Since post-translationally modified proteins and peptides are often present at low stoichiometry level, PTM analyses generally require enrichment steps with established protocols, and newly identified PTMs often need to be manually verified.

Data analysis and visualization provided with personal support to each project

The results are presented in easily interpretable manners according to the chosen workflow. Usually, normalized protein intensities are provided with protein annotation, and differentially regulated proteins are determined based on regulation factors and statistical significance. Publication-quality figures can be generated in house. Functional analysis including GO and pathway analyses and/or other extended analysis can also be provided upon request.