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.

Peptide Resolution by MALDI-TOFC = 12.000

C = 12.011

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

Peptide Matches for PMF of m/z 1529.73 Peptide

Peptide Sequence Identification Matched m/z difference

FQTCDTGKEYPLK expressed pro 1528.722 (0.008)

SVGSDSEFQQISR hypothetical pro 1528.715 (0.015)

TDWSKAPFTASYR Glucanase 1528.73 (0.000)