Lecture25 - Lecture 25: Mass Spectrometry Applications...

Info iconThis preview shows pages 1–3. Sign up to view the full content.

View Full Document Right Arrow Icon
Lecture 25: Mass Spectrometry Applications § Measuring Protein Abundance • ICAT • DIGE § Identifying Post-Translational Modifications § Protein-protein Interactions • Co-Immunoprecipitation Adapted from slides originally created by Dr. James Bruce Proteome Measurement – Protein abundance 1) Isotope-Coded Affinity Tags - ICAT 2) 2D-Difference Gel Electrophoresis - DIGE ICAT Technology § Measures relative abundance of proteins between two samples • e.g., normal cell and cancer cell samples • or proteins isolated from cells grown at normal temp. vs heat shock § Proteins are labeled at cysteine residues with ICAT tags § One sample is labeled with heavy ICAT tag; the other sample is labeled with the light ICAT tag § Heavy and light ICAT tags differ by 8 daltons • Peptides labeled with heavy or light ICAT tag can be distinguished by mass in a mass spectrometry experiment § By comparing abundance of heavy vs light ICAT peptides, one can estimate the relative abundance of a protein in each sample Isotope-Coded Affinity Tags § Different ICAT tags are used to label two different protein samples § One sample is labeled with the heavy ICAT tag (X = deuterium) § Other protein sample is labeled with the light ICAT tag (X = hydrogen) § Heavy vs Light ICAT tags differ in mass by 8 daltons § Biotin is used for affinity purification of ICAT-labeled peptides ICAT Tag: Image from: Shiio and Aebersold (2006) Nature Protocols 1 :139-145
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
ICAT Experimental Strategy 1. React ICAT tags with free thiol groups in proteins (cysteines) • Heavy ICAT tag with Sample 1; Light ICAT tag with Sample 2 2. Mix samples, and digest with protease (e.g., trypsin) to generate peptides 3. Affinity purify ICAT-labeled peptides with Avidin • Biotin moiety in ICAT tag interacts strongly with Avidin 4. Compare relative MS signals for heavy- and light-labeled peptides
Background image of page 2
Image of page 3
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 01/20/2012 for the course MBIOS 478 taught by Professor Staff during the Fall '11 term at Washington State University .

Page1 / 7

Lecture25 - Lecture 25: Mass Spectrometry Applications...

This preview shows document pages 1 - 3. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online