1

I. Introduction

I. Introduction1. Definition: Protein Characterization/Proteomics

i. Classical Proteomicsii. Functional Proteomics

2. Mass spectrometeryI. Advantages in Studying Proteins II. General configuration

2

I. Introduction

Why is proteomics necessary?(Pandey, A., Mann, M., Nature, 405, 837-846, 15June2000)

• complete sequences of genomes is not sufficient to elucidate biological function

• existence of an open reading frame in genomic data doesn’t imply the existence of a functional gene (8% error in annotations for 340 genes from theMycoplasma genitalium genome) verification of gene product is an important first step in genome annotating

• modifications of proteins are not apparent from the DNA sequence (isoforms, post-translational modifications)

• mRNA may or may not correlate with protein level

• localization of gene product can be determined

• protein-protein interaction and molecular composition of cellular structures such as organelles can be determined only at the

protein level

3

I.1. Definition: Protein Characterization/Proteomics

“Proteome” (Wilkins and Williams)• entire protein complement of a given genome

“Proteomics” • naturally: study of the proteome • catalog and characterize these proteins• large scale analysis of proteins within a single experiment (or series thereof)

Proteomics is classified into two disciplines classical proteomics functional proteomics

Protein Characterization• one protein at a time

8

I.2. Mass Spectrometry

Definition/Requirement: Mass Spectrometry

• technique to determine the relative weight of atoms and molecules by separation of charged atoms and molecules based (ions) on their massin the gas phase.(first mass spectrometer 1910, Ne-isotope 20/22)

• molecules need to be in the vapor phase

• molecules need to be ionized

9

I.2.i. Advantages in Studying Proteins

High mass accuracy

• 10 ppm: 1000 Da ± 0.01 Da (UNC)

Identification of proteins via database searching Detection of post-translational modifications

High sensitivity

• femto-mol (=50 pg of 50 kDa protein) (UNC)

Study proteins at physiological level

Provides sequence information

Identification of modification sites

10

I.2.ii. General configuration

• ion source: ionization and transfer ofmolecules into the gas phase

• mass analyzer: separation of the moleculesdue to their mass

ion source

mass analyzer detector

11

II. Mass Spectrometry

II. Mass Spectrometry1. Analytical Parameters/Definitions

i. Molecular weightii. Mass Accuracyiii. Chemical Background vs. Peakiv. Mass Resolution

12

II.1.i. Molecular weight

Mono-isotopic molecular weight:

• mass of the molecule which elementary composition possesses only the most natural abundant isotopes

(12C, 1H,16O,14N, etc.)

Average-isotopic molecular weight:

• calculated mass of the molecule out of a elementary composition possesses isotopes in the proportion corresponding to their natural abundances

13

II.1.i. Molecular weight

Masses and Abundance of isotopes of natural elements:Element O# M# Mass Relative

abundanceAverage mass

C 6 12 12.000000 98.90 12.011

13 13.003355 1.10

H 1 1 1.007825 99.985 1.00794

2 2.014102 0.015

O 8 16 15.994915 99.762 15.9994

17 16.999131 0.038

18 17.999159 0.200

N 7 14 14.003074 99.634 14.0067

15 15.000109 0.366

S 16 32 31.972072 95.02 32.066

33 32.971459 0.75

34 33.967868 4.21

36 35.967079 0.02

14

II.1.i. Molecular weight

Expected mass:Acetic acid: C2H4O2

Isotopes: 12C, 13C, 1H, 2H,16O,17O, 18O

Monoisotopic: 12C21H4

16O2

90 possible formulas6 formulas with significant abundances

composition mass relative abundance12C2

1H416O2 60.02113 100.000

12C13C1H416O2 61.02448 2.224

12C21H4

16O17O 61.02534 0.07612C2

1H32H16O2 61.02741 0.060

12C21H4

16O18O 62.02538 0.40113C2

1H416O2 62.02784 0.012

15

II.1.ii. Chemical Background vs. Peak

Definition:

• peak: must be at least twice the baseline; S/N > 2 • peak: more than one data point is needed to define a peak

• chemical background: chemical must be evaluated to show peak

comes from sample

16

II.1.ii. Chemical Background vs. Peak

Molecular weight peak width increases with mass:Peptide Mass MH+ Rel. abundance

Leu5-enkephalinC28H38N5O7

556.28557.28558.28559.29

100.0034.04

6.890.89

monoisotopic 556.64 average mass

PNGF fragmentC89H140N27O26

2003.052004.052005.052006.052007.052008.06

88.61100.0060.1425.26

7.781.68

monoisotopicbase peak 2004.26

average mass

Ubiquitin C378H630N105O118S

8560.628561.638562.638563.638564.638565.648566.648567.648568.658569.658570.658571.66

4.1419.0247.2778.4798.75

100.0086.5863.6240.3922.09

8.813.19

monoisotopic

base peak8565.89

average mass

17

II.1.iii. Mass Accuracy

Mass accuracy

• mass accuracy = ΔM(calculated-observed)

• in Da, amu, ppm (parts per million)• ppm: [(m/zobs-m/zcalc)/m/z calc] x 106

Mass spectrometer Mass accuracy for peptides

FTICR highest ~ 1-5 ppmTOF high ~ 10-50 ppmIon-trap/quadrupoles moderate/low > 500 ppm

18

II.1.iv. Mass Resolution

Definition:

• Resolution = R = M/ΔM • ΔM : width at half height

• estimating unit resolution: M/0.5

• estimating complete isotopic (M, M’) resolution:([M+M’]/2)/([M-M’] x 0.5)

ΔM

m/z

19

II.1.iv. Mass Resolution

Example acetic acid (see slide 14):

• “unit” resolution:

60 Da and 61 Da R=60/0.5=120

• “complete” isotopic resolution:

61.02448 Da and 61.02534 Da R=61.02491/0.00043=141,918

20

II.1.iv. Mass Resolution

Resolution illustrated: angiotensin II C50H73N13O12

(from K.G. Owens, M.M. Vestling “Fundamentals and Applications of MALDI-TOF-MS”, A Short Course Spnosored by the American Society for Mass Spectrometry)

21

II.1.iv. Mass Resolution

Resolution illustrated: ubiquitin C378H630N105O118S

(from K.G. Owens, M.M. Vestling “Fundamentals and Applications of MALDI-TOF-MS”, A Short Course Spnosored by the American Society for Mass Spectrometry)