1. UPDATE! In-Class Wed Oct 6 Latil de Ros, Derek Buns, John

2. 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)

3. 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 (O 18 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!

4. 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!

6. Bottom-Up versus Top-Down Approaches

7. Two Types of Mass Spectrometers 1. MALDI-ToF (intact peptides) 2. ESI-MS/MS (peptide fragmentation  sequence) SAMPLE MASS ANALYZER DETECTOR ionsresolved 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.

8. 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

9. 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.

10. 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

11. Using Mass Spec Data to Identify Proteins www.matrixscience.comwww.matrixscience.com, and select “MASCOT”

12. Identify the protein from which these tryptic peptides were derived

13. 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.

14. SCROLL DOWN

15. Entered values

16. 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)!

17. 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

18. Peptide Matches for PMF of m/z 1529.73 Peptide Peptide SequenceIdentification Matched m/z difference FQTCDTGKEYPLKexpressed pro1528.722 (0.008) SVGSDSEFQQISRhypothetical pro1528.715 (0.015) TDWSKAPFTASYRGlucanase1528.73 (0.000)

19. 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

20. Tandem Mass Spec (MS/MS) Common Fragmentation Methods: - collisions with gas - infrared laser - electron capture MS-I 

21. Fragmentation along the peptide backbone in MS/MS

23. 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.

26. Membrane proteins tend to have alkaline pI values