SILAC

Stable Isotope Labeling with Amino acids in Cell culture (SILAC) is a quantitative proteomic approach which allows the comparison of protein levels in up to 3 cultured cell extracts. With this approach it is possible to identify and quantitate thousands of proteins in a single experiment. SILAC-labelled samples sent to the facility are fractionated and analysed by nano-LC MSMS using our Orbitrap Fusion Tribrid mass spectrometer.

A straightforward metabolic labeling technique used for MS-based quantitative proteomics, SILAC allows the identification and quantitation of thousands of proteins in a single experiment. In this approach, samples under comparison are differentially labeled and then mixed prior to analysis by nano-LC MSMS.

Typically, SILAC uses lysine and arginine, which in combination with tryptic digestion result in the labeling of every peptide in the sample (with the exception of the C-terminal peptide of each protein). A standard SILAC experiment can be used to compare two or three samples by labeling as follows:

• Light label (K0R0): Cells grown in normal media with natural amino acids.
• Medium Label (K4R6): Cells grown in media containing ²H₄-lysine and ¹³C₆-arginine.
• Heavy label (K8R10): Cells grown in media containing ¹⁵N₂¹³C₆-lysine and ¹⁵N₄¹³C₆-arginine.

The SILAC method relies on the complete incorporation of heavy amino acids during protein turnover. The heavy amino acids create a distinct and known mass difference between the samples. However, since labeling does not affect the chemical properties of the peptides derived from each sample, they co-elute from the LC column and are analysed together in the mass spectrometer. The peptide peaks of the differentially labeled samples can be very accurately quantified relative to each other to determine the peptide ratios. Furthermore, protein quantitation is based on the median values of multiple peptides identified from the same protein, resulting in highly accurate protein quantitation between samples.

TMT

Tandem Mass Tagging (TMT) is a quantitative proteomic approach which allows the comparison of protein levels in up to 10 different samples in a single experiment. It is applicable to any sample type. With this approach it is possible to identify and quantitate thousands of proteins in a single experiment. Samples for TMT analysis are labelled within the facility, fractionated and analysed by nano-LC MSMS using the SPS-MS3 approach on our Orbitrap Fusion Tribrid mass spectrometer.

The TMT reagents label peptides prepared from any sample type and allow the identification and quantitation of thousands of proteins in a single experiment.

The TMT reagents are available in 2-plex, 6-plex and 10-Plex formats, allowing the comparison of multiple samples in a single experiment. The samples to be analysed are digested and then labeled with the TMT reagents, a set of amine-specific isobaric tags, yielding differentially labeled peptides which are identical in mass due to mass distribution within the tag between a reporter group and a mass normaliser. Each of the reporter groups has a defined mass. The labeled samples are then pooled and fractionated using basic RP chromatography, Strong Anion eXchange (SAX) chromatography or Strong Cation eXchange (SCX) chromatography. Each fraction is then analysed by acidic RP nano-LC MSMS using our Orbitrap Fusion Tribrid mass spectrometer. Fragmentation of the isobaric tag releases the low molecular mass reporter ions which are used to quantify the peptides. Protein quantitation is based on the median values of multiple peptides identified from the same protein, resulting in highly accurate protein quantitation between samples.

The use of SPS-MS3 for accurate TMT-based quantitation

During a TMT analysis of complex samples two main factors contribute to decreased quantitative accuracy when using conventional MS2 analysis; interference in reporter ion mass regions and interference from co-isolation of precursor ions. These factors have been shown to mask subtle but biologically significant changes in abundance levels.

Interference from background ions in the reporter region can be addressed by scanning with high resolution and co-isolation interference has recently been addressed by using MS3 analysis. However, although the MS3 workflow has been demonstrated to markedly improve quantitative accuracy when analysing complex samples, for most instruments this is at the expense of the number of observed quantified peptides and proteins. This is due in part to the scan rate of the instrument but more importantly to the fact that only one product ion from the MS2 scan can be selected for further fragmentation (MS3). The Orbitrap Fusion mass spectrometer, the instrument used for TMT analysis in our Proteomics facility, offers a higher scan rate and a novel SPS (synchronous precursor selection) MS3 technology in which up to 20 MS2 product ions are isolated simultaneously and undergo MS3. Consequently, the Orbitrap Fusion is able to generate unsurpassed accuracy in quantitation without the reduction in the number of proteins identified which is associated with MS3 analysis on other instruments.