Mass Spectrometry - Introduction - Ion sources & sample introduction - Mass analyzers - Basics of biomolecule MS - Applications

Adapted from Mass Spectrometry in Biotechnology Gary Siuzdak, Academic Press 1996
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Mass Spectrometry
Introduction
- In the past, mass spectrometry was confined to the realm of small molecules; large molecules did not survive the desorption and ionization process intact. - Matrix-assisted laser desorption/ionization (MALDI). - Electrospray ionization (ESI) - Fast atom/ion bombardment (FAB) - The analysis of proteins, peptides, carbohydrates, oligonucleotides, natural products and drug metabolites. - Detection capabilities ranging from the picomole to the femtomole level. - Molecular weight accuracy on the order of 0.01%. - The utilities of MS include molecular weight characterization, noncovalent interactions, protein and peptide sequencing, DNA sequencing, protein folding, in vitro drug analysis, and drug discovery.
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Mass Spectrometry
Introduction

m/z

Light source

Prism Light detector

Mass Spectrometry
- Matrix-assisted laser desorption/ionization
- A nonvolatile solid as the matrix.

Mass Spectrometry
- Matrix-assisted laser desorption/ionization

Mass Spectrometry
- Electrospray ionization

Mass Spectrometry
- Electrospray ionization

Mass Spectrometry
- Electrospray ionization myoglobin z1 x 1542 = M + z1 (z1-1) x 1696 = M + (z1-1) z1 z1 = 11 M = 16,951 z1-1

Mass Spectrometry
Mass analyzers

Quadrupole Time-of-flight & time-of-flight reflectron Ion trap Magnetic Double-focusing magnetic sector FT-IR (ion cyclotron
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Mass Spectrometry
Mass analyzers
Resolution is the ability of a mass spectrometer to distinguish between ions of different mass-to-charge ratios. Therefore, greater resolution corresponds directly to the increased ability to differentiate ions. Resolution = M / M = M1 / (M1 M2) M = full width at half maximum (FWHM)

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Mass Spectrometry
- Quadruple analyzer

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Mass Spectrometry
- Time-of flight analyzer & time-of-flight reflectron

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Mass Spectrometry
- Tandem mass spectrometry

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Mass Spectrometry
- Tandem mass spectrometry

ABC DEF ABC DEC ABC DBF ABC DEF

-I
mass ABC only scan all masses scan all masses mass ABC only

AB, BC ABC AB, DE C, etc. AC, DF AB, etc. AB, BC ABC*

-II
scan all masses mass C only mass minus B mass BC only

AB BC ABC

Daughter Scan

Parent Scan

DEC ABC DF AC BC

Parent Scan

SRM

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Mass Spectrometry

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Mass Spectrometry
Basics of biomolecules mass spectrometry
- 1. Salt content should be minimized. 2. Matrix selection/preparation. 3. Care should be taken to maintain high purity. 4. Sample solubility in the solvent or matrix is crucial. 5. Is there enough amount of sample? 6. The functional groups help to determine how to analyze. 7. Analyze soon after synthesis and/or purification.

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Mass Spectrometry
Basics of biomolecules mass spectrometry Solubility: 14-kDa protein electrospray mass analyzed
methanol

33% methanol 33% acetonitrile 33% water

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Mass Spectrometry
Basics of biomolecules mass spectrometry MALDI matrix

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Mass Spectrometry
Applications

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Mass Spectrometry
Applications Protein folding

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Mass Spectrometry
Overview of protein identification

} TagIdent

- Primary attributes & Secondary attributes. - Most attributes relate directly or indirectly to a proteins sequence, however they vary in the way that they are generated and the protein property they represent. - Crossed references. Adapted from Micromass and Proteome Research: New Frontiers in Functional Genomics

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Mass Spectrometry
ESI- Tandem MS MALDI-TOF MS

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Mass Spectrometry
Sample Preparation for Mass Spectrometric Analysis
- Pure proteins or protein spots from 1-D or 2-D gel electrophoresis - Protein digestion with proteases to yield fragments that are most compatible with MS analysis, 6 - 20 amino acids (i.e. Trypsin: a 50kDa to 30 tryptic peptides). - Non-specific protease and CNBr. - Extraction of peptides from gel spots. - Exact masses of the resulting peptides. - Fragmentation of peptide ions in MS analysis. - 5 - 10 fmol level for peptide mapping. - 50 - 100 fmol level for peptide sequencing.
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Mass Spectrometry
Peptide mass fingerprinting (PMF) or mapping

Trypsin *

* * *

* *

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Mass Spectrometry
Peptide mass fingerprinting (PMF) or mapping

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Mass Spectrometry
Peptide mass fingerprinting (PMF) or mapping using MASCOT

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Mass Spectrometry
Peptide fragmentation to generate partial sequence

Sequencing

Fragmentation

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Mass Spectrometry
Selection of charged peptides

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Mass Spectrometry
Transformation & de-isotoping of raw MS-MS data.

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Mass Spectrometry
Sequencing results from MS-MS of m/z 841.5 (M+3H)3+

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Mass Spectrometry
Sequence reading

L = I, Q/K = G+A, W = G+E = A+D

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Mass Spectrometry
Sequencing results from MS-MS of m/z 841.5 (M+3H)3+ -Galactosidase

Q (89%) versus G (9%) A (4%) at position 3 W (35%) versus G (65%) E (65%) at position 18 G (80%) E (80%) versus A (19%) D (19%) at position 21
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Mass Spectrometry
Peptide sequencing using MASCOT

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Mass Spectrometry
Peptide sequencing using MASCOT

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Mass Spectrometry
Protein identification by peptide sequencing

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Mass Spectrometry
Peptide sequencing using chemical modifications lysine to homoarginine
Partial sequencing plus MALDI-TOF

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Mass Spectrometry
Amino acid residue modifications

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Mass Spectrometry
Chemical modifications

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Mass Spectrometry
Post-translational modifications

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Mass Spectrometry
Quantitative analysis using isotope-coded affinity tags (ICAT)

Nature biotechnology (1999) 17:994-999

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