Mass Spectrometry. Mass spectrometer MALDI-TOF ESI/MS/MS. Basic components. Ionization source Mass analyzer Detector
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1 Mass Spectrometry MALDI-TOF ESI/MS/MS Mass spectrometer Basic components Ionization source Mass analyzer Detector 1
2 Principles of Mass Spectrometry Proteins are separated by mass to charge ratio (limit 1 charge/1.5-2kda) Charge occurs through ionization process Most common ionization methods in proteomics Matrix assisted laser desorption ionization (MALDI) Electro-spray ionization (ESI) ESI Advantages Samples are in solution Small sample volumes and sizes Can be coupled to HPLC (nano-hplc or UHPLC) Can be run in both positive and negative mode Results in multiple charging so larger proteins can be measured Disadvantages Not all molecules will ionize High maintenance Only uses small fraction of the sample 2
3 ESI ESI 3
4 Multiple charging of Proteins Cytochrome C Relative Abundance m/z Deconvoluted Data / Relative Abundance mass 4
5 Determining Ion Charge Charge is calculated from the separation of the peaks in a resolved isotope series MALDI gives singly charge ions (usually) ESI gives multiply charged ions SINGLY CHARGED Δ m/z, amu DOUBLY CHARGED Δ m/z, amu MALDI Samples are mixed with a matrix and placed on the surface of a target Target is placed inside the vacuum of MS Samples are ionized by high energy laser Most/all samples ionize Usually single charge 5
6 MALDI MALDI target Matrix from syringe Peptides from LC column mixer Moving stage Matrix + peptides spotted on MALDI target 6
7 Common Matrices for Protein MALDI Peptide Abbrev. Compound Structure Mass < 10 kda CHCA α-cyano-4-hydroxy cinnamic acid Mass > 10 kda SA Sinapic acid HABA 2-(4-Hydroxyphenyl azo)benzoic acid THAP 2,4,6-Trihydroxy Acetophenone DHB 2,6-Dihydroxyaceto Phenone Mass Analyzers Quadrapole Time of Flight (TOF) Ion Trap Fourier Transformed Ion Cyclotron 7
8 Quadrapole Time of Flight 8
9 Ion Trap Nature Reviews Drug Discovery 2, (February 2003) ltq_animation_ Fourier transformed ion cyclotron FTICR_WMKeck_NCSU video of FTICR and how it works no sound 9
10 Mass spectrometers So how do we use these? Full mass Mass of complexes Peptide map Sequencing for identification Quantification 10
11 MALDI-TOF Peptide Map Sequencing Process Protein digested with protease Typically trypsin which cleaves at K and R Peptides separated by HPLC (nano-hplc) Analyzed by MS/MS Several problems exist De novo sequencing difficult Fragments may be too large or not sufficiently charged Poor ionization of fragments Post translational modifications 11
12 1. Sample is injected into reverse phase HPLC and peptides separated. 2. Fragments are separated by mass in first quadrapole mass analyzer 3. Selected ions enter second quadrapole analyzer and mixed with argon to fragment peptides. 4. Daughter ions are analyzed by TOF mass spectrometer. MS sequencing Peptide Sequence b ions S G F L E E D E L K y ions 100 % Intensity m/z 12
13 Fragmentation of Peptides Amino Acid Masses Amino acid Mass(avg) Amino acid Mass(avg) G D A Q S K P E V M T H C F I R L Y N W
14 Ambiguous Masses Amino acid combination Mass (amu) Single amino acid Acetylated amino acid Mass (amu) Unmodified amino acid G-G 114 N Ac-G 99 V G-A 128 K/Q Ac-A 113 L/I V-G 156 R Ac-S 129 E G-E 186 W Ac-N 156 R A-D 186 W S-V 186 W How do we deal with this? Use available information The genome Edman sequences Comparison to known proteins Use programs such as Protein Prophet, Sequest, Mascot 14
15 Sequencing with MS/MS Currently two main search engine programs are used to identify sequences rather than creating the sequence from the data. SEQUEST (Xcor values > 1.9, 2.2, or 3.7 for ions of 1, 2, or 3 charges are usually accurate) Mascot (Scores of >40-50 give good assignments) This process requires that the peptide be from a protein that the sequence is known. From an organism with a sequenced and anotated genome. Protein was purified and sequenced. Present in an EST library. Has identity or high similarity with a protein from another organism. Quantification by MS SILAC (stable isotope labeling of amino acids in cell culture) In vivo labelling with C 13 or N 15 ICAT (Isotope coded affinity tag) itraq (Isobaric tag for relative and absolute quantitation) Competing technology DIGE (Differential Gel Electrophoresis) 15
16 SILAC ICAT-label 4 parts to ICAT molecule A protein reactive group Iodocetamide covalently links to free cysteines. An affinity tag biotin concentrates the cysteine-containing peptides, reducing complexity. 16
17 ICAT-label An isotopically labeled linker (C 10 H 17 N 3 O 3 ) The linker chain can substitute up to nine 13C atoms. The light and heavy molecules are chemically identical Comparison of labeled peptides provides a ratio of the protein concentration in the original sample. An acid cleavage site: remove biotin and part of the linker by adding TFA. reduces the mass of the tag improves the overall peptide fragmentation efficiency. itraq Label up to 8 samples at once Amine specific labeling (lysine and N- terminal) Mass of all labels the same After fragmentation reporter labels are found between m/z 113 and m/z 119 Ratio of peaks of reporter ions is proportional to relative concentrations. 17
18 Sensitivity of Different Methods Silver stain below 1 ng (linear 1-60 ng) Colloidal coomassie blue 100 ng/protein spot Deep Purple <1 ng Sypro ruby 1 ng (linear ng) DIGE ng (linear ng-10 μg) MS 500 fm FTICR MS 500 am For a 60 kda protein 500 fm = 30 ng 500 am = 30 pg 18
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