Differential Proteomics
The general workflow of proteomics starts with digesting a mixture of 10’s of micrograms of proteins into peptides with a specific protease (e.g., using trypsin), which are separated using nano flow liquid chromatography and determining the mass-to-charge
(m/z) of the tryptic peptides in a mass spectrometer (Figure 1). In a standard proteomics experiment, tandem MS/MS is used to fragment peptides. We then apply bioinformatics to identify the sequence of individual peptides and quantitate
them and use this information to infer protein abundances. There are several approaches to quantify differences between proteins within samples. Label-free quantitation (LFQ) is our most common method to determine protein abundance (orange box, Figure 2):
An equal concentration of peptides is loaded onto the column. Relative quantitation is performed by comparing the extracted peak intensity of a given peptide across runs in the dataset. Samples can be multiplexed using stable-isotope- labeling with
amino acids in cell culture (SILAC) in your laboratory, where “light” or “heavy” versions of specific amino acids are metabolically incorporated into samples in cell culture, which are mixed in equal ratios and processed. Intensities
of peptide extracted ion chromatograms from the MS1 scan can be used to quantify relative protein abundances between samples. Additional isobaric labeling multiplexing strategies such as TMT, each sample is digested into peptides, labeled with a unique
isobaric label, and mixed in equal ratios. During MS/MS analysis, each tag yields a fragment with a unique fragment mass that can be used for relative quantitation. For absolute quantitation, targeted MS is used. Select peptides of proteins of interest
are individually monitored and quantified using a doped-in heavy labeled internal standard (IS) peptide. Informatics are used to identify global differences as well as proteins and pathways that are significantly altered between analysis groups. The
MSF has experience in proteomics analyses of samples from cells, tissues, tumors, exosomes, secretomes, and exposomes. The number of proteins identified are experiment and cell-type dependent and ranges from 2,000-7,000 proteins for
90 min LCMS runs.