Upload
rosamund-moody
View
230
Download
0
Tags:
Embed Size (px)
Citation preview
In ClassMon Oct 4 Thu Oct 7 Mon Oct 11
Deter, Rebekah Haider, Waseem Han, JenniferNiziolek, Olivia Hall, Pam Rong, MaRavanlou, Ali Koester, Bob
Siddappaji, Madhu Lopez Nicora, HoracioSiebers, Matt Quarles, Devin
Yang, Hui-Ching VanBuren, RobertWest, Ellen Wang, Zuguang
Zehr, Brian
Schedule of Extra-Credit Talks
7-9PM, 138 ERML
Lecture 2.
Basics of 2-DE and MALDI-ToF MS
1. Difficulties of Proteomics
2. Tools of proteomics
3. Basic types of mass spectrometers
4. Shotgun proteomics
5. MALDI and peptide mass fingerprinting
Inherent Difficulties with Proteomics
NUCLEIC ACIDS
• Similar properties
• Relatively stable
• PCR amplification
• Microarrays: predictable hybridization
PROTEINS
Tools of Proteomics
1. Protein separation techniques (2-DE)
2.Mass spectrometry
3.Databases (genome/transcriptome analysis)
4. Algorithms to match MS data to proteins
5. Specific proteases (known cut sites)
Comparative 2-DE Analysis of Tomato Root Proteins
Figure 3. Rose et al. Plant J. 39: 715
Protein Separation Techniques: 2-DE
Copyright ©2003 American Society for Biochemistry and Molecular Biology
Steel, L. F. (2003) Mol. Cell. Proteomics 2: 262-270
2DE of total human serum proteins and proteins fractionated on anti-HSA immunoaffinity resin
Enzyme and cleavage rules
Enzyme or Reagent
Cleaves where?
Exceptions
Trypsin (higher specificity)
C-terminal side of K or R
if P is C-term to K or R; after K in CKY, DKD, CKH, CKD, KKR; after R in RRH, RRR, CRK, DRD, RRF, KRR
Lys CC-terminal side of K
Asp NN-terminal side of D
Glu C (bicarbonate)
C-terminal side of E
if P is C-term to E, or if E is C-term to E
Glu C (phosphate)
C-terminal side of D or E
if P is C-term to D or E, or if E is C-term to D or E
ALLOW 1 MISSED CLEAVAGE (1 MC)!
‘Classic’ Combination of 2-DE and MALDI-ToF MS
Spot Picker
Digest with trypsin
“PMF”
MALDI-Tof-MS
Sample
Two Types of Mass Spectrometers
1. MALDI-ToF (intact peptides)
2. ESI-MS/MS (peptide fragmentationsequence)
SAMPLE MASS ANALYZER
DETECTOR
ions resolved ions
(MALDI) (TOF)
LASER
For a peptide of mass 1032, addition of a proton increases the mass to 1033.
m/z = 1033 for the [M+H]+ ion in MALDI.
Isotopes
+Most elements have more than one stable isotope.
For example, most carbon atoms have a mass of 12 Da, but in nature, 1.1% of C atoms have an extra neutron, making their mass 13 Da.
+Why do we care?
Mass spectrometers can “see” isotope peaks if their resolution is high enough.
If an MS instrument has resolution high enough to resolve these isotopes, better mass accuracy is achieved.
How do mass spectrometers get their names?
Types of ion sources:
• Electrospray (ESI)
• Matrix Assisted Laser Desorption Ionization (MALDI)
Types of mass analyzers:
• Quadrupole (Quad, Q)
• Ion Trap
• Time-of-Flight (TOF)
-Either source type can work with either analyzer type: “MALDI-TOF,” “ESI-Quad.”
-Analyzers can be combined to create “hybrid” instruments. ESI-QQQ, MALDI QQ TOF, Q Trap
ExPASy Proteomics Server (UniProt)http://www.uniprot.org
Search and retrieve a specific protein
Use ‘PeptideMass’ to cut with specific proteases in silico.
Predicting Proteolytic Fragments
Peptide Mass Fingerprint Searches: PROWL(ProFound)http://prowl.rockefeller.edu/ select ProFound
Effect of Mass Accuracy and Mass Tolerance on PMF Search Results (ARATH)
Search m/z Mass tolerance (Da) # Hits
1529 1 >100
1529.73 0.01 84
1529.734 0.001 10