Proteins (in-solution or separated on SDS-PAGE gels) are processed for enzymatic digestion (reduction, alkylation, digestion typically with trypsin) and resulting peptides are analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). For peptide identification, database search software tools score matches between the aquired MS2 spectra and in silico produced spectra extracted from a protein sequence database. Based on the peptide sequences, the protein identity is infered.
Our spatial proteomics protocol integrates the precision of a laser capture microdissection (LCM) microscope with the power of mass spectrometry-based proteomics. This approach enables the highly specific isolation of microscopic regions of interest, followed by in-depth protein discovery and characterization. To maximize performance, we have adapted our protocol for low-input samples, ensuring reliable analysis even when material is scarce. Additionally, we leverage the data-independent acquisition (DIA) method, which enhances proteome coverage and quantification reproducibility, enabling robust and unbiased protein profiling. This combination provides unprecedented insights into the spatial distribution and functional roles of proteins, particularly in challenging or heterogeneous biological samples. For more details about our spatial proteomics workflow, please visit this page.
Following protein identification, the identification and localization of post-translational modifications is also possible. Popular modifications include phosphorylation, acetylation, ubiquitination and methylation but also additional modifications can be assessed (upon discussion with the staff). Dedicated software tools provide information with possible modification sites that in most times have to be verified manually to ensure confident results. Due to the stoichiometry, PTMs detection can be challenging therefore specific sample enrichment steps or custom developed methods could be considered.
It can be performed in a variety of manners.
SILAC-based: Stable isotope labelling by amino acids in cell culture (SILAC) is a method to study the relative proteomic changes between two or three different treatments. The methodology relies on the metabolic incorporation of amino acids with substituted stable isotopic nuclei. In SILAC, a given ‘light’ “medium” or ‘heavy’ form of the amino acid is incorporated into three samples. The three cell populations are grown in culture media that are identical except that one of them contains a ‘light’ and the others contain a “medium” or ‘heavy’ form of a particular amino acid (e.g. 12C and 13C labeled L-lysine and arginine). As the isotopically labeled amino acids are chemically identical, their incorporation does not interfere with normal cell growth but the proteins/peptides are distinguishable by mass. Upon completion of labelling (which is monitored by the facility), equal numbers of cells are mixed together and processed for mass spectrometry-based identification. Relative quantitation occurs at peptide level, by comparing the “light” “medium” and “heavy” peptide peak areas. In most cases, a peptide-level fractionation step prior to MS analysis is recommended to reduce sample complexity thus increase the proteome coverage.
TMT-based: Tandem Mass Tag (TMT) chemical labelling is a technique that utilizes isobaric reagents to label the primary amines of peptides enabling the simultaneous protein identification and relative quantitation of up to 11 different samples in the same experiment. Samples to be quantified are independently prepared and lysed to extract proteins. Proteins are digested, peptides are labelled with different TMT reagents and equal amounts of labelled peptides are mixed. The combined peptide mixture is analyzed by LC-MS/MS for identification and quantitation. Quantitation is based on the relative intensities of the reported ions in the MS2 spectra. In most cases, a peptide-level fractionation step prior to MS analysis is recommended to reduce sample complexity thus increase the proteome coverage.
Label-free : Labe-free quantitation (LFQ) is based on peptide precursor area comparison across independently analyzed samples. All samples are processed and run on the mass spectrometer individually.
Proteins rarely act in isolation but rather interact with other molecules to function. Protein-protein interactions govern a broad range of cellular processes such as signal transduction and communication. Mass spectrometry-based proteomic approaches play a pivotal role in deciphering these interaction networks which have an intrinsic temporal and spatial heterogeneity. From conventional Affinity Purification (AP-MS) to quantitative proximity labeling (BioID and APEX), the past two decades have witnessed a rapid progress enabling not only to decipher protein-protein interaction networks but also study the interaction dynamics.
Targeted proteomic assays such as selected reaction monitoring (SRM; also referred as multiple reaction monitoring, MRM) can be used for sensitive quantitation of selected proteins. The quantitation is typically performed by comparing the peak areas of transitions to those of internal standards such as heavy isotope-labeled synthetic peptides providing high specificity, precision and accuracy. Moreover, SRM assays can be multiplexed, are able to distinguish modified and unmodified forms of the protein, and can be developed with high success rates.
The presentation of the large amounts of obtained data in easily interpretable manner poses a great challenge. In-housed developed solutions are used for producing high quality plots for data visualization. Additionally, training and consulting on using freely available tools can be provided.