UPDATE!

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

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

• derivatization (O18 labeling)

• enzymatic digestion of minute amounts of protein prior to mass spec.

• quick

• small volumes

• 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:

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.

A. Spot intensity on 2-DE gels

B. ICAT labeling of peptides, ETC!

Bottom-Up versus Top-Down Approaches

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.

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

Resolution =

Peak mass

Half max peak width

(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.

C = 12.000

C = 12.011

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 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)!

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 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.

Ion source

MS-2MS-1

Mixture of ions

Single ion

Fragments

Tandem Mass Spec (MS/MS)

Common Fragmentation Methods:- collisions with gas- infrared laser- electron capture

MS-I

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