Improving peptide throughput and accuracy of results
Stable isotope labeling with amino acids in cell culture (SILAC) is a powerful and widely used method for identifying and quantifying relative changes in complex protein samples. It can be applied to complex biomarker discovery and systems biology studies as well as to isolated proteins and protein complexes.
SILAC involves the labeling of protein samples in vivo by substituting an isotopically heavy form of an amino acid for the naturally occurring light form. Because labeled and unlabeled samples are combined during the initial steps of sample preparation, SILAC minimizes the quantitative error inherent in handling separate samples in parallel. In addition, the mixing of samples permits a variety of enrichment techniques, including immunoprecipitation. These techniques can improve the detection of abundance changes for both low-abundance proteins and posttranslational modifications such as phosphorylation or glycosylation.
Reverse phase separation on Thermo Scientific™ low flow systems can be seamlessly integrated into label-free quantitative workflows and combined with Thermo Scientific Orbitrap mass spectrometers. The Thermo Scientific EASY-nLC 1200 System and UltiMate 3000 RSLCnano System are the LC systems of choice for label-free quantitative proteomics. The EASY-nLC 1200 is designed for operational simplicity and high performance, while the UltiMate 3000 RSLCnano offers versatility and precision.
The Thermo Scientific Orbitrap Fusion Lumos Tribrid Mass Spectrometer features the highest resolving power (up to 500,000 @ m/z 200) for the most accurate, precursor-based SILAC quantitation without compromising acquisition rate. Also, peptide identification is not restricted by fragmentation type; HCD, CID, ETD, and EThcD methods can be used separately or in combination. Because generated fragmentation data are often complementary, the use of multiple fragmentation approaches results in a higher level of peptide identification.
Precursor ion-based quantitation techniques like SILAC require mass spectrometers that provide HRAM without compromising scan speed and spectral quality. The Thermo Scientific Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer combines an HRAM, ultra-high-field Orbitrap analyzer with high performance quadrupole precursor ion selection to deliver unsurpassed speed while maintaining the sensitivity needed for SILAC experiments. System resolutions of up to 240,000 enable discrimination of co-eluting isobaric ions that can interfere with accurate quantitation.
In SILAC, proteins are identified using accurate-mass precursor information combined with HCD, CID, ETD, and/or EThcD fragmentation data. The relative peak intensities of multiple distinct peptides derived from each protein are averaged to determine their overall relative abundance (and fold change) between experimental and control conditions. Peptide SILAC ratios can be calculated using the Precursor Ions Quantifier node and Quantification Method included within the Thermo Scientific Proteome Discoverer Software. Custom quantification methods can also created by editing the SILAC duplex and triplex methods.