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Lecture

Protein Quantification & Identification in Mass Spectrometry

In course

CH-419: Protein mass spectrometry and proteomics

In systems biology, proteomics represents an essential pillar. The understanding of protein function and regulation provides key information to decipher the complexity of living systems. Proteomic technology now enables deep quantitative proteome mapping v ...

Description

This lecture covers the challenges in quantifying proteins using mass spectrometry, including the impact of ionization efficiency on peak intensity and the complexities in measuring analyte abundance. It also discusses techniques like label-free quantification, SILAC, and TMT for comparing peptide levels between samples. The lecture delves into the principles of spectral counting, AQUA peptides for absolute quantification, and the use of Super SILAC mix as an internal standard. Additionally, it explores the role of UniprotKB in providing functional information on proteins and the process of MS/MS ion search using peak lists. The importance of mass tolerances, enzyme selection, and accurate database searching is emphasized.

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Instructor

Loïc Dayon

Official source

Ontological neighbourhood

Chemistry

Analytical chemistry: Mass spectrometry

Related concepts (32)

Mass spectrometry

Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures. A mass spectrum is a type of plot of the ion signal as a function of the mass-to-charge ratio.

Electron ionization

Electron ionization (EI, formerly known as electron impact ionization and electron bombardment ionization) is an ionization method in which energetic electrons interact with solid or gas phase atoms or molecules to produce ions. EI was one of the first ionization techniques developed for mass spectrometry. However, this method is still a popular ionization technique. This technique is considered a hard (high fragmentation) ionization method, since it uses highly energetic electrons to produce ions.

Tandem mass spectrometry

Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyse chemical samples. A common use of tandem MS is the analysis of biomolecules, such as proteins and peptides. The molecules of a given sample are ionized and the first spectrometer (designated MS1) separates these ions by their mass-to-charge ratio (often given as m/z or m/Q).

Electrospray ionization

Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized. ESI is different from other ionization processes (e.g. matrix-assisted laser desorption/ionization (MALDI)) since it may produce multiple-charged ions, effectively extending the mass range of the analyser to accommodate the kDa-MDa orders of magnitude observed in proteins and their associated polypeptide fragments.

Ionization

Ionization (or ionisation) is the process by which an atom or a molecule acquires a negative or positive charge by gaining or losing electrons, often in conjunction with other chemical changes. The resulting electrically charged atom or molecule is called an ion. Ionization can result from the loss of an electron after collisions with subatomic particles, collisions with other atoms, molecules and ions, or through the interaction with electromagnetic radiation.

Related lectures (32)

Mass Spectrometry: Ion Sources and Analyzers

On Mass Spectrometry explores ionization principles, mass sorting techniques, and various mass analyzers.

Mass Spectrometry: Analyzing Peptides with Tandem MS

Covers mass spectrometry and tandem MS/MS techniques for peptide analysis, focusing on ionization, fragmentation, and sequence identification.

Mass Spectrometry: Detectors and Techniques

Explores mass spectrometry detectors, MS/MS, and various techniques for ion detection and analysis.

Proteomics Profiling Methods

Explores proteomics profiling methods, including ABPP, MS approaches, and quantitative target identification using SILAC and TMT labeling.

Mass Spectrometry: Techniques and Applications