Merck
CN
HomeApplicationsProtein BiologyProtein Mass Spectrometry

Protein Mass Spectrometry

Protein mass spectrometry for protein identification, characterization, and quantitation

Protein mass spectrometry is widely used to analyze biological samples for biomarker discovery, proteomics research, and clinical applications. Compared to other techniques used for the large-scale characterization of proteins, mass spectrometry has become a primary tool for proteomics based on its amenability to complex analysis.

Mass spectrometry is used to quantitatively identify and characterize proteins based on their structure, post-translational modifications, and interactions.

  • Protein identification typically involves chemical or enzymatic digestion of proteins into peptides, which are then analyzed by mass spectrometry and identified using computational methods or sequencing.
  • Post-translational modifications can be identified through changes in amino acid residue mass. Sites of modification can be mapped using sequencing or computational methods.
  • For glycan analysis and profiling, enzymatic or chemical methods are used to release glycan moieties from glycoproteins, followed by derivatization of released glycans for mass spec analysis.
  • Protein interactions are determined by affinity co-purification of a specific target protein with any interacting proteins or studied more globally using size exclusion or ion exchange chromatography prior to analysis by mass spectrometry.

Related Technical Articles

  • Mass Spectrometry
    Learn more about Mass Spectrometry or MS including what it is, what it is used for and how it works.
  • Absolute Quantification of Serum Proteome
    Absolute Quantification (Protein-AQUA™™) is a targeted quantitative proteomics technique that exhibits robust efficacy and is being increasingly utilized for a wide variety of quantitative proteomics studies.
  • Protein AQUA™ Applications
    Utilize the protein AQUA™ technique to measure expression of silenced genes, quantitate protein levels for low abundance serum proteins and analyze phosphorylated peptides enriched with immobilized metal affinity chromatography.
  • UPS1 & UPS2 Proteomic Standards
    Standardize Your Research. We offer both the Universal Proteomics Standard and the Proteomics Dynamic range Standard as complex, well-defined, well characterized reference standards for mass spectrometry.
  • Development of a Simple and Rapid Digestion Protocol for Proteomics Sample Preparation
    In this study, we developed a rapid trypsin digest kit that, at elevated temperatures, yielded reliable, reproducible results in less than 2 hours on a wide variety of substrates for mass spectrometry.
  • See All (22)

Related Protocols

For quantitative proteomics, proteins or peptides can be labeled chemically with stable isotopes using tandem mass tagging (TMT) and iTRAQ or metabolically through incorporation of labeled amino acids (SILAC). Comparison of heavy and light isotope incorporation enables relative quantification by correlating mass spec peak intensities to protein abundancies. For absolute quantitation, samples can be spiked with isotopically labeled synthetic peptide or protein standards for selected reaction monitoring (SRM) analysis

In protein mass spectrometry, the masses of different proteins and peptides are determined by measuring the m/z (mass-to-charge) ratio of their gas-phase ions. Mass spectrometers first convert protein molecules into gas-phase ions using an ion source. Next, a mass analyzer separates ionized analytes based on m/z ratio. A detector then records the number of ions at each m/z value. MALDI and electrospray ionization (ESI) are commonly used to ionize peptides or proteins.

MALDI-TOF Mass Spectrometry

MALDI is an ionization method that uses a laser energy-absorbing matrix to generate ions with minimal fragmentation of protein molecules. The sample is first mixed with a suitable matrix material. Next, a pulsed laser irradiates the sample, triggering ablation and desorption of both the sample and matrix material. Analyte molecules are then ionized by protonation or deprotonation in the ablated gases prior to analysis by mass spectrometry.

Electrospray Ionization Mass Spectrometry

Electrospray ionization (ESI) produces ions using an electrospray in which a high voltage is applied to the liquid sample to create an aerosol, generating ions with minimal fragmentation of peptides and proteins. Electrospray ionization is commonly used when coupling mass spectrometry to liquid chromatography (LC-MS), as the liquid chromatography eluate can be fed directly to an electrospray for tandem processing.

Workflow

Sample preparation for protein analysis by mass spectrometry

Protein sample preparation

Protein sample preparation for mass spectrometry requires cell lysis or protein solubilization and stabilization. For proteomic analysis, cells are lysed using buffers that break down the cell membrane with protease inhibitors to prevent protein degradation. High-abundance proteins are removed as necessary. Recovery and enrichment of select fractions facilitate proteomic analysis.

Site-specific proteolysis of samples for protein mass spectrometry

Site-specific proteases, such as trypsin, are used to cleave proteins into small fragments to enable identification by matching experimental spectra to theoretical spectra from protein databases or comparing to run standards. For relative quantitation, cell cultures can be metabolically labeled using stable isotopes incorporated through amino acid feeding or samples can be labeled with stable isotopes using chemical methods. Spiking samples with isotopically labeled synthetic peptides permits absolute quantitation through selected reaction monitoring (SRM) analysis.

Use of calibrators and standards for protein mass spectrometry

Calibration standards can serve as controls for sample analysis and be used to determine protein identity, experimental sensitivity, digestion efficiency, and aid in chromatographic separation and quantitative analysis.

Use of tandem chromatography in protein mass spectrometry

Chromatography allows separation of proteins and peptides into more manageable samples for analysis. Because multiple distinct peptides can have similar masses, HPLC is commonly used to prevent the simultaneous addition of peptides with very similar or identical masses to the mass spectrometer, increasing the overall dynamic range of measurements.

Protein detection and analysis in mass spectrometry workflows

Proteins and peptides are ionized by MALDI or ESI prior to detection and analysis. A mass analyzer distinguishes ions based on their m/z values. Resulting fragmentation patterns can be used for identification, and samples can be quantitated relatively by evaluating peak intensity ratios from samples differentiated by isotopic labeling or quantitated absolutely using SRM analysis with labeled internal standards.

Related Applications
Lysis & Protein Extraction
An overview of cell lysis and protein extraction methods including detergent solubilization, freeze-thaw lysis, osmotic shock, sonication, enzymatic cell lysis, and mechanical disruption techniques such as Dounce, Polytron, and mortar and pestle homogenization.
Protein Labeling & Modification
Protein labeling applications using enzyme-protein labeling, fluorescent protein labeling, and targeted protein degradation technologies.
Protein Expression
Protein expression technologies for expressing recombinant proteins in E. coli, insect, yeast, and mammalian expression systems for fundamental research and the support of therapeutics and vaccine production.
Protein Structural Analysis
Protein structure provides valuable information that can be used to infer protein function. The study of protein structure and mapping of protein interactions, expression levels, and location enables the identification of disease biomarkers and potential drug targets for therapeutic treatment.
Protein Pull-Down
Pull-down assays, reagents, and protocols for investigating in vitro protein-protein interactions using affinity or GST pull-down, tandem affinity purification (TAP), and co-immunoprecipitation methods.
Large Molecule HPLC
High performance liquid chromatography (HPLC) can be used to separate and identify different large biomolecules such as protein and peptides in a sample. It is based on the pumping of a sample with a solvent (mobile phase) through a column packed with sorbent material (stationary phase) at a high pressure.
Cell & Developmental Biology Research
Rooted in physiology and biochemistry, cell and development biology features an interdisciplinary approach to understand intracellular and extracellular dynamics.
Protein Quantitation
Protein quantification methods, reagents, and immunoassay technology for accurately measuring the protein concentrations in a variety of samples.

Highlights

Mass spectrometry workflow and tools
ISOTEC®

Primer for stable isotope labeling by amino acids in cell culture (SILAC)

Download PDF
Primer for stable isotope labeling by amino acids in cell culture (SILAC)
Tools for Mass Spectrometry

Mass spectrometry workflow and tools to monitor proteins by mass spectrometry

Download PDF
Sign In To Continue

To continue reading please sign in or create an account.

Don't Have An Account?