Quantitative proteomics workflows, methods, and resources for advanced insights

Quantitative proteomics workflows, methods, and resources for advanced insights

Proteomics has evolved from a qualitative technique to a continuum of qualitative and quantitative using highly sensitive and accurate mass spectrometry to gain significant biological insights that span a wide range of applications, including biology, biochemistry, biomarker discovery and precision medicine.

Quantitative proteomics using mass spectrometry (LC-MS) allows system-wide identification and quantification of proteins for both discovery-based (untargeted) proteomics applications and targeted proteomics applications. With less sample than western blotting and no antibodies needed, modern quantitative mass spectrometry analyses can detect and quantify thousands of proteins in a single experiment across multiple conditions, and the dynamics can be studied at a level that provides much greater understanding of how biological processes respond to different stimuli, or how they change within a cell, tissue, or organism over time, or while in a disease state.

Thermo Fisher Scientific offers solutions for quantitative mass spectrometry to meet the diverse needs of labs, including TMT quantification, label-free quantitation, SILAC quantitation and SureQuant targeted quantitation.

As part of an end-to-end proteomics workflow solution, the Thermo Scientific Orbitrap Astral Mass Spectrometer extends the dynamic range of accurate quantitative measurements at a faster throughput and with deeper coverage. 

Contact us  Download brochure  More about Orbitrap Astral MS

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Quantitative proteomics review: Magic of multiplexing in LC-MS

Learn how the power of quantitative proteomics and multiplexing technology like tandem mass tag (TMT) mass spectrometry allows understanding of how proteins make a cell functional, why and how molecules move about in a cell and practical applications.


How MS based proteomics overcome issues and limitations of older quantitative proteomics methods

Western blotting is a traditional method used for quantitative protein measurement, but it requires knowledge of the system and expected changes in order to obtain an appropriate target antibody. Antibodies are not always available, not specific, or they’re expensive, and for posttranslational modifications they can be even more difficult to obtain. In addition, western blotting is sample intensive, it has limited linear dynamic range, and typically only a single target is quantified in each western blot.

Modern quantitative proteomics methods such as liquid chromatography coupled to mass spectrometry (LC-MS) can measure changes in the abundance of protein-specific posttranslational modifications (PTMs), facilitating location of the modified residue. Compared to the western blotting method, quantitative proteomics mass spectrometry analyses require less sample, no antibodies, and can detect and quantify multiple proteins in a single experiment across multiple conditions.

Traditional quantitative proteomics methodsIssues and limitations of older proteomics methodsBenefits and capabilities of MS based proteomics
  • Western blotting
  • Enzyme-linked immunosorbent assays (ELISAs)
  • Antibody dependent
  • Sample intensive
  • Time intensive
  • Limited linear dynamic range
  • One target per western blot
  • Assay development time
  • Limited sensitivity in complex matrices
  • No antibodies needed
  • Small or large sample sizes
  • Broad dynamic range
  • Single or multiple samples measured
  • Multiple proteins identified and quantified
  • Highly accurate and sensitive
  • Faster time to result

Orbitrap LC-MS technology is advancing quantitative proteomics

Triple Quadrupole LC-MS and Orbitrap LC-MS technology is advancing quantitative proteomics

Our mass spectrometers created and defined the benchmark in proteomics during the 90s, identifying and cataloging proteins in a proteome or biological system. This remains the foundation of proteomics, but to understand biology, we knew we had to go beyond identification. With continued advancements in sensitivity, dynamic range and throughput, our instruments are helping researchers capture more complete profiles to achieve a new benchmark which includes quantitation. We understand the scientific community needs to understand the functions of individual proteins, protein complexes, and their place in complex biological systems, and easily translate protein abundance changes into valuable discoveries. In addition to our leading instrumentation, we’re committed to and invested in developing fit-for-purpose reagents, software and workflows to meet the high demands of modern proteomics.

Choosing the most appropriate quantitative proteomics technique depends on experimental demands and instrumental capabilities. We offer multiple mass spectrometers for quantitative proteomics experiments plus a full range of products and resources to assist you along the entire workflow. Regardless of which technique you decide is best, know that we’re here to help, so never hesitate to contact one of our technical experts for assistance as you make decisions about how to approach your work.

InstrumentIdeal use based on application Supported workflowsResources

Orbitrap Astral Mass Spectrometer
With an unprecedentedly low detection level, the Orbitrap Astral Mass Spectrometer accesses more proteins from a single cell than ever before. And with the ability to multiplex single cells at a higher throughput, hundreds of single cells can be quantitatively analyzed in a single day, allowing researchers to efficiently and reliably determine the heterogeneity of a cell’s biology, unlocking never-before-seen insights that can revolutionize research.

  • LFQ
  • TMT
  • SIM
  • DIA
  • SureQuant
  • DDA
  • AIF
Orbitrap Eclipse
Orbitrap Ascend Tribrid Mass Spectrometer
Obtain maximum insights on your most complex molecules and biological systems, from whole proteome profiling and quantitation, structural characterization to multiplexed single-cell proteomics. With new innovations that deliver the ultimate flexibility in experimental scope, it accelerates your path to new, impactful results, so you can drive your science beyond today’s discovery.
Q Exactive Plus
Orbitrap Exploris 480 Mass Spectrometer
Obtain maximum quantitative insights from untargeted proteome profiles to targeted proteomics experiments with industry leading single-cell sensitivity and extraordinary accuracy, precision and simplicity. With curated workflows that deliver greater usability, it accelerates your path to large-scale studies, delivering proven high data quality and time savings, so you can go beyond faster to actionable outcomes.

Orbitrap Exploris 240 Mass Spectrometer
Orbitrap Exploris 240 Mass Spectrometer
Expand your capabilities from small- to large-scale studies across a variety of applications from protein identification, quantitation, to multiplexing proteomics studies. With optimized methods, it delivers a fast turnaround of sample to results with operational simplicity. Best-in-class performance, all within a compact footprint, so you can go beyond with everyday versatility.

Quantitative proteomics applications, workflows and product solutions

Quantitative discovery proteomics using mass spectrometry seeks to identify and characterize as many proteins as possible across a broad dynamic range while also measuring the relative protein abundance changes happening in multiple sample sets. This is also referred to as untargeted proteomics experiments.

View the workflow from sample preparation to mass spectrometry and data analysis, plus our recommended products for each step by clicking on the links in the table below.

 Tandem Mass Tag (TMT) QuantitationStable Isotope Labeling with Amino Acids in Cell Culture (SILAC)Label-Free Quantitation (LFQ)
 High-throughput multiplexed protein quantification with a highly sensitive and advanced workflow using TMT label reagents, LC-MS instrumentation and proteomics data software which enables relative quantitation of more samples simultaneously.Proteins labeled in vivo substituting an isotopically heavy form of an amino acid for the naturally occurring light form, followed by HRAM Thermo Scientific Orbitrap MS analysis and peptide calculation software for accelerated, accurate identification and relative quantification.Relative quantitation of protein samples from any origin, tested and analyzed individually with high performing LC-MS instruments and then evaluated and interpreted with scale using the advanced label-free quantitation node within our Thermo Scientific Proteome Discoverer software.
Samples per LC-MS1-161-31
Precision (%CV)<5-10<10-15<10-20
AccuracyVery goodGoodGood
  • Applicable to any sample type
  • Multiplexing increases MS throughput
  • Least susceptible to inter-sample variations in sample handling and preparations
  • Multiplexing increases MS throughput
  • Applicable to any sample type
  • Cost-efficient sample
  • Minimal sample handling
  • Requires extensive fractionation or long chromatographic gradients
  • Only readily applicable to cell cultures
  • Increases MS spectral complexity
  • Each sample runs individually (low throughput)
  • Requires very reproducible LC separations
  • Requires multiple technical replicates
 Workflow & ProductsWorkflow & ProductsWorkflow & Products

On-demand webinar

Quantitative approaches in discovery proteomics: How do they measure up?

Get an overview of the current state-of-the art techniques utilized in the field of quantitative discovery proteomics plus a review of the most common workflows and approaches, featuring a comparative study which objectively evaluates the performance of different quantitative approaches using the latest mass spectrometry instrumentation and analysis tools. This webinar will help participants set reasonable expectations and will help in the selection of appropriate workflows according to experimental objectives

Watch webinar

Quantitative targeted proteomics using mass spectrometry is used to determine relative or absolute abundances of peptides representing the proteins of interest with a high degree of accuracy and sensitivity. Frequently applied to large sample sets, and often used on target peptides selected through analysis of data from earlier discovery experiments, it allows profiling of hundreds of targets in a single experiment.

 SureQuant Internal Standard (IS) Targeted QuantitationParallel Reaction Monitoring (PRM)Selected Reaction Monitoring (SRM)Selected Ion Monitoring (SIM)
Key benefitSensitivity and efficiencyHigh selectivityHighest sensitivityFlexibility
 A new paradigm for absolute quantitation of target proteins building on the PRM approach, enabling reliable detection and quantification of hundreds to thousands of targets in a wide range of samples while maintaining speed, sensitivity, and overall performance.Utilizing the built-in Orbitrap mass analyzer in our newest instruments, it detects all target product ions in parallel using one, concerted high resolution mass analysis, most suitable for quantifying tens to hundreds of targets in complex matrices.Performed on a triple quadrupole, it’s the gold standard for reliable, more routine, targeted quantitation, relative or absolute, and is ideally suited for analyzing and quantifying large numbers of samples.Isolates a selected peptide ion characteristic of the targeted protein; only the selected target is transferred to the analyzer for detection, no fragmentation, simple method set up, most suitable for quantifying tens of proteins in samples of medium complexity.
Samples per LC-MS1111
Precision (%CV)<5-10<5-10<5-10<5-10
AccuracyVery goodVery goodVery goodVery good
  • Highest target multiplexing capabilities in a single targeted analysis
  • Enhanced acquisition efficiency for higher throughput
  • Reduced assay development time
  • High selectivity
  • High sensitivity
  • Eliminates most interferences
  • Fast method setup
  • Enables confident confirmation of peptide identity with spectral library match
  • High sensitivity
  • High dynamic range
  • High reliability for large sample volume
  • Uses same MS system as discovery quantitation
  • Option to multiplex, isolating up to ten targets
  • Increases sensitivity 5- to 50-fold compared to full-scan MS
  • Higher up front cost of labeled peptides
  • Requires reproducible LC separations
  • Limited number of targets
  • Assay development time
  • Limited sensitivity in complex matrices
  • Requires reproducible LC separations
  • Limited sensitivity in complex matrices
 Workflow & products   

Label-free quantitation (LFQ): Explore the proteome in greater depth

As a cost-effective alternative to isotopic labeling approaches, label-free quantitation enables relative quantitation of protein samples from any origin. Samples are tested individually using advanced software with chromatographic features that align the various runs. The biggest advantage of this approach is that the number of sample comparisons is not limited. Additionally, peptide identification can be performed with any fragmentation method (CID, ETD, EThcD, and/or HCD).

TMT Quantitation: Achieve high-throughput multiplexed protein quantitation

Proteomics techniques are evolving to become a highly sensitive, quantitative, and high-throughput approach to analyzing global protein dynamics within a cell, tissue or an organism. In the past decade, critical advances have been made across the entire proteomics workflow, most notably the development of Tandem Mass Tag Reagents and state-of-the-art advancements in mass spectrometry, which have enabled more accurate, high-throughput (comparison of up to 18 samples in a single mass spectrometry analysis) quantitative proteomics.

  • Unlimited numbers of samples can be compared
  • Samples can originate from any source
  • Meticulous handling and preparation required for accuracy and reproducibility of complex samples
  • Samples quantified in the same scan
  • Mixing samples following digestion and labeling permits a variety of fractionation and enrichment techniques
  • Kits available for studying up to 16 samples in complex analysis with multiple conditions (e.g. time, dose, replicates, etc.)
  • No extensive sample preparation
  • Can be time consuming because samples have to be run one at a time
  • Wide selection of kits available with all the necessary reagents for preparing and comparing 2-16 samples can save time
  • Labeling takes time
  • Cost effective for mass spectrometer owners
  • Cost of kits and labeling materials
  • Cost effective for non-instrument owners

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