Quantitative proteomics strategies using Tandem Mass Tags (TMT) enablable sample multiplexing and precise measurement of protein abundance. However, co-isolated ion interference can suppress accurate ratio quantification. Employing MS3 methods with synchronous precursor selection (SPS) on Orbitrap Tribrid mass spectrometers can minimize ion interference. Therefore, we developed a standardized commercially available TMT11plex yeast digest standard to detect co-isolation interference and enable MS method optimization.
Here, we utilized TMT11plex to label peptides from four strains of Saccharomyces cerevisiae; a parental line and three lines respectively lacking the non-essential protein Met6, His4, or Ura2. Tryptic peptides from the strains lacking gene MET6, HIS4, OR URA2 were labeled in triplicate, while the parental line was labeled in duplicate.
We demonstrate that a TMT11plex yeast digest standard can be used as a proteomic reference standard to measure protein/peptides identification and optimize acquisition and data analysis methods to limit co-isolation interference, as well as diagnosis MS instrument status by monitoring mass accuracy, ion injection time, and reporter ion signal to noise. We then used the standard to establish a standardized workflow including two LC methods (50min or 120min gradients) for a variety of nano-spray liquid chromatography setups including Easy-nLC 1200 and 1000, and Dionex U3000, optimized MS acquisition settings for Hybrid or Tribrid Orbitrap mass spectrometers, and data analysis in Proteome Discoverer 2.2. The TMT11plex yeast digest standard provides mass spectrometry users a tool to measure the accuracy, precision, and dynamic range assessments for different mass spectrometry approaches, and is an excellent quality control assay to the assessment of the LC and MS instrument status when combined with a standardized workflow.