2. Basics of 2-DE and MALDI-ToF MS
Lecture 2.
Basics of 2-DE and MALDI-ToF MS
Difficulties of Proteomics
Tools of proteomics
Basic types of mass spectrometers
Shotgun proteomics
MALDI and peptide mass fingerprinting

4. Inherent Difficulties with Proteomics
NUCLEIC ACIDS
Similar properties
Relatively stable
PCR amplification
Microarrays: predictable hybridization
PROTEINS

5. Tools of Proteomics Protein separation techniques (2-DE)
Mass spectrometry
Databases (genome/transcriptome analysis)
Algorithms to match MS data to proteins
Specific proteases (known cut sites)

6. Protein Separation Techniques: 2-DE
Comparative 2-DE Analysis of Tomato Root Proteins
Figure 3. Rose et al. Plant J. 39: 715

7. 2DE of total human serum proteins and proteins fractionated on anti-HSA immunoaffinity resin
Steel, L. F. (2003) Mol. Cell. Proteomics 2:
Copyright ©2003 American Society for Biochemistry and Molecular Biology

8. 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 C
C-terminal side of K
Asp N
N-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)!

9. ‘Classic’ Combination of 2-DE and MALDI-ToF MS
Sample
Digest with trypsin
Spot Picker
MALDI-Tof-MS
“PMF”

10. Two Types of Mass Spectrometers
MALDI-ToF (intact peptides)
ESI-MS/MS (peptide fragmentationsequence)
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.

11. 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.

12. Peptide Resolution by MALDI-TOF
C =
C =

13. 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

14. Predicting Proteolytic Fragments
ExPASy Proteomics Server (UniProt)
Search and retrieve a specific protein
Use ‘PeptideMass’ to cut with specific proteases in silico.

15. Peptide Mass Fingerprint Searches: PROWL(ProFound)
select ProFound

16. Effect of Mass Accuracy and Mass Tolerance on PMF Search Results (ARATH)
Search m/z Mass tolerance (Da) # Hits
>100

17. Peptide Matches for PMF of m/z 1529.73 Peptide
Peptide Sequence Identification Matched m/z difference
FQTCDTGKEYPLK expressed pro (0.008)
SVGSDSEFQQISR hypothetical pro (0.015)
TDWSKAPFTASYR Glucanase (0.000)