UPDATE!
Schedule of Extra-Credit Talks
7-9PM, 138 ERML
In Class
Mon Oct 4
Thu Oct 7
Mon Oct 11
Deter, Rebekah
Niziolek, Olivia
Ravanlou, Ali
Siddappaji, Madhu
Siebers, Matt
Yang, Hui-Ching
West, Ellen
Haider, Waseem
Hall, Pam
Koester, Bob
Lopez Nicora, Horacio
Quarles, Devin
VanBuren, Robert
Wang, Zuguang
Zehr, Brian
Han, Jennifer
Rong, Ma
In-Class Wed Oct 6
Latil de Ros, Derek
Buns, John
Lecture 6.
Mass Spec Basics─continued
1. Bottom Up versus Top Down
2. MS versus MS/MS
3. Using MASCOT to identify proteins
4. Importance of pI (isoelectric point)
1. Know what terms mean; e.g., “O- and N-linked glycosylation”
2. Question 5: Describe the essence of the ‘reactor’ they developed.
• (pre)concentration
• quick
• derivatization (O18 labeling)
• small volumes
• enzymatic digestion of
minute amounts of protein
prior to mass spec.
• cost effective
3. Similar strategies can be applied to other PTMs and difficult proteins!
Some Key Concepts
1. Top Down vs Bottom Up vs Shotgun approaches
2. Two basic types of mass spectrometers: MS and MS/MS
3. All mass spectrometers measure m/z, but z can vary!
4. Mass spec per se is not quantitative. Therefore must use
other approaches to get quantitative information:
A. Spot intensity on 2-DE gels
B. ICAT labeling of peptides, ETC!
5. Enrichment allows detection of low abundance proteins or
low stoichiometry PTMs. (e.g., Zhou et al. paper)
6. Accuracy of measurement matters!
7. Genomic sequence (and deduced protein sequences) are
essential to mass spec approaches.
Bottom-Up versus Top-Down Approaches
Two Types of Mass Spectrometers
1. MALDI-ToF (intact peptides)
2. ESI-MS/MS (peptide fragmentationsequence)
ions
LASER
SAMPLE
(MALDI)
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.
resolved ions
MASS
ANALYZER
DETECTOR
(TOF)
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)
• Fourier transform ion cyclotron resonance (FT-ICR)
-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
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-TOF
C = 12.000
Resolution =
Peak mass
Half max peak width
C = 12.011
(e.g., 1245/045 = 2766)
FOR MOST APPLICATIONS, THE EFFECTIVE MAXIMUM
SIZE OF A PEPTIDE THAT FLIES IN MALDI IS BETWEEN
500 and 3,000 Da. PEPTIDE SIZE MATTERS.
Using Mass Spec Data to Identify Proteins
www.matrixscience.com, and select “MASCOT”
Identify the protein from
which these tryptic
peptides were derived
Note that only 5 of the 6 queried
peptide masses matched. If the
‘unmatched list’ was longer you
could rerun it without the matched
masses.
Click here to see which peptides
matched and coverage.
SCROLL DOWN
Entered values
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)!
Predicting Proteolytic Fragments
ExPASy Proteomics Server (UniProt)
http://www.uniprot.org
Search and retrieve a specific protein
Use ‘PeptideMass’ to cut with specific
proteases in silico.
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)
What is MSMS?
MS/MS means using two mass analyzers (combined
in one instrument) to select an analyte (ion) from a
mixture, then generate fragments from it to give
structural information.
Mixture of
ions
Ion
source
Single
ion
MS-1
Fragments
MS-2
Tandem Mass Spec (MS/MS)
MS-I 
Common Fragmentation
Methods:
- collisions with gas
- infrared laser
- electron capture
Fragmentation along the peptide
backbone in MS/MS
Protein Isoelectric Points—who cares?
1. Protein Identification:
• pH extremes often difficult
• Observed values should reasonably match
the predicted pI
2. Post-translational Modification (PTM):
• Can affect pI—”beads on a string”
3. Interesting correlation between pI and subcellular
compartment.
Membrane proteins tend to have alkaline pI values