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Expanding experimental scale and increasing sample throughput
Translational research focused on identifying and verifying biomarker panels demands an efficient and effective LC-MSn workflow solution to expand the experimental scale and increase sample throughput while quantifying with greater specificity.
The Thermo Scientific Stellar Mass Spectrometer creates a new paradigm for targeted quantitative solutions for large and small molecules that enables unprecedented laboratory productivity.
Superior quantitative performance at scale to confidently quantify more analytes per unit time
“The Stellar mass spectrometer will enable robust targeted quantitation on a scale beyond most people’s imagination.”
Michael J. MacCoss
Professor of Genome Sciences
University of Washington
Translational research, moving from discovery to verification to leverage targeted quantitation, can identify large numbers of putative biomarkers. To leverage increased sensitivity and quantitative accuracy, LC-MS/MS workflows must have acquisition speed and real-time data management. The Stellar Mass Spectrometer utilizes full scan MS2 acquisition with hyper-fast acquisition speeds (up to 140 Hz) and Adaptive RT for dynamic retention time adjustments ensuring reproducible measurements with sensitivity and specificity to achieve comprehensive sample coverage.
Initial studies created targeted quantitation from extracellular vesicles originally evaluated in an untargeted discovery experiment. A total of 8,686 tryptic peptide surrogates, representing nearly 2,000 proteins, were reproduced and precisely quantified in 60 minutes. The quantified proteins were evaluated using Reactome pathway mapping to evaluate signal transduction pathways. The large protein set enabled evaluation of multiple pathways in one experiment.
Fabregat A, et al. Reactome diagram viewer: data structures and strategies to boost performance. Bioinformatics (Oxford, England). 2018 Apr;34(7) 1208-1214. doi: 10.1093/bioinformatics/btx752. PubMed PMID: 29186351. PubMed Central PMCID: PMC6030826.
Faster data acquisition speeds are needed to address expanding experimental scale
Targeted metabolite quantitation presents challenges for translational researchers who leverage large-scale study sizes to determine biology. To improve sample throughput, faster chromatographic gradients are used for targeted quantitative analysis of endogenous metabolites and their isotopically enriched standards aiming for enhanced quantitative accuracy and method standardization. Targeting heavy/light pairs increases target capacity per unit time and reduces the duty cycle, potentially compromising quantitative performance.
With Stellar Mass Spectrometer’s fast acquisition speed, it can deliver high-quality quantitative data, even for 1-second-wide peak widths when using capillary electrophoresis. The results show quantitative accuracy and precision for 124 compounds in less than 2 minutes covering a wide concentration range (1-500 µM) as well as align with high accuracy against the NIST reference standard.
Overcome the challenges of quantitative biomarker analysis in non-depleted plasma proteomics
The PQ500 Peptide Reference kit was spiked into non-depleted plasma digest across a dilution series ranging from 0.003 to 100 fmol on column. Targeted-MS2 scan events were created for both the 799 heavy labeled and corresponding endogenous (light) peptides. The Stellar Mass Spectrometer achieved precise quantitation (CV ≤ 20%) on 96.6% of the heavy labeled peptides with a median CV of 4.72% as compared to a previous publication showing precise quantitation of 97.9% of the heavy labeled peptides with a median CV of 6.1% using 2x longer gradient while only targeted the heavy labeled peptides.
The linear quantitation curve shows the accuracy of the heavy-to-light area value ratios across the spiking range. The inset shows comparative XICs for the heavy (blue) and light (red) tMS2 results at the 1 fmol spiking level. Despite the low intensity levels and narrow peak width, 7 data points were still acquired for both peptide analogues for confident quantitation of the peptide ELLDTVAPQK down to ca. 0.033 fmol on column.
Single-cell sensitivity with robust quantitation
Targeted protein quantitation performed on the Stellar mass spectrometer increases quantitative sensitivity down to zeptomole levels and improves sample throughput.
The Stellar Mass Spectrometer performs fast tMS2 acquisition to expand the targeted peptide list covering more than 300 proteins that can be critical to cell development or other key biological functions.
Sting functional association networks search for PUF60 network and corresponding coverage from the introductory experiment as represented by the proteins circled in red.
Increase selectivity and specificity to extend quantitative dynamic range
Phosphoproteins represent signaling nodes for key cellular pathway functions that are analyzed to determine biological responses or perturbations. Targeted quantitation of site-specific phosphorylation within the phosphoprotein can measure activity based on the occupancy rate.
Even with IMAC or TiO2 enrichment, the relative abundance of targeted phosphopeptides to matrix peptides can compromise the quantitative effectiveness of tMS2 acquisition. The Stellar Mass Spectrometer can leverage PRM Conductor software to intelligently design tMS3 acquisition methods to acquire fast Synchronous Precursor Selection MS3 (SPS MS3) data with incredible sensitivity increasing the value of the targeted phosphopeptide experiment.
Platform versatility expedites method creation for bile acid analysis
Method optimization for small molecules often requires determination of critical tandem MS parameters per analyte to achieve sensitive and specific data acquisition. This is time consuming and can be limited by one type of dissociation mechanism.
The Stellar Mass Spectrometer’s fast polarity switching and MSn acquisition speed enables comprehensive analyte characterization in one or two LC-MSn experiments, instead of hours or days. Acquiring beam-type HCD as well as on-resonance CID fragmentation can generate alternative quantitative strategies, bypassing the need to use a less-specific SIM quantitation. Validated tandem MS spectra can also be imported from the Thermo Scientific mzCloud Mass Spectral Library for confirmation.
Leverage fast tMS3 acquisition for quantitation of challenging lipid biomarkers
Triacylglycerides (TAG), critical lipid classes involved in metabolism, are quantified for health assessment. Isomeric TAGs often co-elute using standard chromatographic separation methods requiring MS3 fragmentation to introduce the specificity needed to quantify each isomer.
The Stellar Mass Spectrometer acquires fast tMS2 and tMS3 data up to 140 and 40 Hz, respectively, enabling qualitative and quantitative TAG analysis in one experiment.
Solutions for additional research support
For Research Use Only. Not for use in diagnostic procedures.