- Introduction - Ion sources & sample introduction - Mass analyzers - Basics of biomolecule MS - Applications Adapted from Mass Spectrometry in Biotechnology Gary Siuzdak,, Academic Press 1996 1
Introduction Mass Spectrometry - In the past, mass spectrometry was confined to the realm of small l molecules; large molecules did not survive the desorption and ionization process intact. - Matrix-assisted 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 e level. - Molecular weight accuracy on the order of 0.01%. - The utilities of MS include molecular weight characterization, noncovalent n interactions, protein and peptide sequencing, DNA sequencing, protein folding, in vitro drug analysis, and drug discovery. 2
Introduction Mass Spectrometry m / z Light source Prism Light detector 3
- Matrix-assisted assisted laser desorption/ionization - A nonvolatile solid as the matrix. 4
- Matrix-assisted assisted laser desorption/ionization 5
- Electrospray ionization Mass Spectrometry 6
- Electrospray ionization Mass Spectrometry 7
- Electrospray ionization myoglobin Mass Spectrometry z 1 x 1542 = M + z 1 (z 1-1) x 1696 = M + (z 1-1) z 1 z 1-1 z 1 = 11 M = 16,951 8
Mass analyzers Mass Spectrometry - Quadrupole - Time-of of-flight flight & time-of of-flight flight reflectron - Ion trap - Magnetic - Double-focusing magnetic sector - FT-IR (ion cyclotron 9
Mass analyzers Mass Spectrometry Resolution is the ability of a mass spectrometer to distinguish between ions of different mass-to to-charge ratios. Therefore, greater resolution corresponds directly to the increased ability to differentiate ions. Resolution = M / M M = M 1 / (M 1 M 2 ) M M = full width at half maximum (FWHM) 10
- Quadruple analyzer Mass Spectrometry 11
- Time-of flight analyzer & time-of of-flight flight reflectron 12
- Tandem mass spectrometry 13
- Tandem mass spectrometry 離子源 質譜儀 -I 解離室 質譜儀 -II 質譜圖 子離子掃瞄 Daughter Scan ABC DEF mass ABC only AB, BC ABC scan all masses AB BC ABC 母離子掃瞄 Parent Scan ABC DEC scan all masses AB, DE C, etc. mass C only DEC ABC 中性丟失掃瞄 Parent Scan ABC DBF scan all masses AC, DF AB, etc. mass minus B DF AC 選擇反應偵測 SRM ABC DEF mass ABC only AB, BC ABC* mass BC only BC 14
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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. 16
Basics of biomolecules mass spectrometry Solubility: 14-kDa protein electrospray mass analyzed methanol 33% methanol 33% acetonitrile 33% water 17
Basics of biomolecules mass spectrometry MALDI matrix 18
Applications Mass Spectrometry 19
Applications Mass Spectrometry Protein folding 20
Overview of protein identification } TagIdent - Primary attributes & Secondary attributes. - Most attributes relate directly or indirectly to a protein s s 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 21
ESI- Tandem MS MALDI-TOF MS 22
Sample Preparation for Mass Spectrometric Analysis - Pure proteins or protein spots from 1-D 1 D or 2-D 2 D gel electrophoresis - Protein digestion with proteases to yield fragments that are most t 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. 23
Peptide mass fingerprinting (PMF) or mapping Trypsin * * * * * * * 24
Peptide mass fingerprinting (PMF) or mapping 25
Peptide mass fingerprinting (PMF) or mapping using MASCOT 26
Peptide fragmentation to generate partial sequence Sequencing Fragmentation 27
Selection of charged peptides 28
Transformation & de-isotoping of raw MS-MS data. 29
Sequencing results from MS-MS of m/z 841.5 (M+3H) 3+ 30
Sequence reading L = I, Q/K = G+A, W = G+E = A+D 31
Sequencing results from MS-MS of m/z 841.5 (M+3H) 3+ β-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 32
Peptide sequencing using MASCOT 33
Peptide sequencing using MASCOT 34
Protein identification by peptide sequencing 35
Peptide sequencing using chemical modifications lysine to homoarginine Partial sequencing plus MALDI-TOF 36
Amino acid residue modifications 37
Chemical modifications 38
Post-translational translational modifications 39
Quantitative analysis using isotope-coded affinity tags (ICAT) Nature biotechnology (1999) 17:994-999 40