Mass Spectrometry MALDI-TOF ESI/MS/MS Mass spectrometer Basic - PDF document

11/29/2012 Mass Spectrometry MALDI-TOF ESI/MS/MS Mass spectrometer • Basic components – Ionization source – Mass analyzer – Detector 1

11/29/2012 Principles of Mass Spectrometry • Proteins are separated by mass to charge ratio (limit 1 charge/1.5-2kDa) • Charge occurs through ionization • Most common ionization methods in proteomics – Matrix assisted laser desorption ionization (MALDI) – Electro-spray ionization (ESI) Electro-Spray Ionization 2

11/29/2012 ESI • Advantages – Samples are in solution – Small sample volumes and sizes (  l/min) – 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 Multiple charging of Proteins Charge size est MW +12 17 727.50 12367.50 16 772.40 12358.40 1031.0 100 15 825.00 12375.00 Cytochrome C +13 14 884.00 12376.00 +11 951.8 13 951.80 12373.40 1124.6 12 1031.00 12372.00 +8 11 1124.60 12370.60 1545.7 10 1236.90 12369.00 9 1374.20 12367.80 +14 +10 8 1545.70 12365.60 Relative Abundance 7 1766.60 12366.20 884.0 1236.9 Avg 12369.23 50 +9 Stdev 4.77 1374.2 +7 1766.6 +15 825.0 +17 +16 727.5 772.4 0 1000 2000 200 m/z 3

11/29/2012 Deconvoluted Data 12369 +/- 2 100 Relative Abundance 50 0 12500 15000 10000 mass 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 DOUBLY CHARGED SINGLY CHARGED  1  0.5 4

11/29/2012 Matrix Assisted Laser Desorption Ionization • 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 MALDI 5

11/29/2012 Mass Analyzers • Quadrapole • Time of Flight (TOF) • Ion Trap • Fourier Transformed Ion Cyclotron Mass Spectrometry Basics + Heavy + pole ions - 2000V +1 +1 +2 +2 Light Ions - pole 6

11/29/2012 Quadrapole http://www.chemistry.adelaide.edu.au/external/soc-rel/content/quadrupo.htm http://www.youtube.com/watch?feature=endscreen&v=WbX27Gg5ziU&NR=1 http://hk.youtube.com/watch?v=8AQaFdI1Yow&NR=1 Time of Flight 7

11/29/2012 Ion Trap Nature Reviews Drug Discovery 2 , 140-150 (February 2003) http://www.youtube.com/watch?v=3uUwa1DDoHQ http://www.youtube.com/watch?v=KjUQYuy3msA&feature=related Fourier transformed ion cyclotron resonance www.pnl.gov/news/release.asp?id=249 FTICR_WMKeck_NCSU http://hk.youtube.com/watch?v=a5aLlm9q-Xc&feature=related video of FTICR and how it works no sound 8

11/29/2012 Using MS Data • So how do we use these? – Full mass – Mass of complexes – Peptide map – Sequencing for identification – Quantitation MALDI-TOF Peptide Map 9

11/29/2012 Protein 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 is very difficult – Fragments may be too large or not sufficiently charged – Poor ionization of fragments – Post translational modifications MS sequencing 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. 10

11/29/2012 Fragmentation of Peptides http://www.matrixscience.com/help/fragmentation_help.html Peptide Sequence 88 145 292 405 534 663 778 907 1020 1166 b ions S G F L E E D E L K 1080 1166 1022 875 762 633 504 389 260 147 y ions 100 % Intensity 0 m/z 250 500 750 1000 11

11/29/2012 Amino Acid Masses Amino acid Mass(avg) Amino acid Mass(avg) G 57.0520 D 115.0886 A 71.0788 Q 128.1308 S 87.0782 K 128.1742 P 97.1167 E 129.1155 V 99.1326 M 131.1986 T 101.1051 H 137.1412 C 103.1448 F 147.1766 I 113.1595 R 156.1876 L 113.1595 Y 163.1760 N 114.1039 W 186.2133 Ambiguous Masses Amino acid Mass Single Acetylated Mass Unmodified combination amino acid amino acid amino acid (amu) (amu) G-G 114.104 N Ac-G 99.09 V 114.1039 99.1236 G-A 128.1308 K/Q Ac-A 113.1225 L/I 128.1308 113.1595 128.1742 V-G 156.1378 R Ac-S 129.1219 E 156.1876 129.1155 G-E 186.1675 W Ac-N 156.1509 R 186.2133 156.1876 A-D 186.1674 W 186.2133 S-V 186.2108 W 186.2133 12

11/29/2012 Closely Related Sequences ATSARA1A pI = 6.10 ATSARA1b pI = 6.52 MW = 21952.33 MW = 21972.45 MFLFDWFYGI LASLGLC K KE AKILFLGLDN AGKTTLLHML ATSARA1A MFLFDWFYGI LASLGLW Q KE AKILFLGLDN AGKTTLLHML ATSARA1B KDERLVQHQP TQHPTSEELS IGKI N FKAFD LGGHQIARRV ATSARA1A KDERLVQHQP TQHPTSEELS IGKI K FKAFD LGGHQIARRV ATSARA1B WKD C YAKVDA VVYLVDAYDR DRF V ESKREL DALLSDEALA ATSARA1A WKD Y YAKVDA VVYLVDAYDK ERF A ESKREL DALLSDEALA ATSARA1B N VP C LILGNK IDIPYA S SED ELRY Y LGLTN FTTGKG I VNL ATSARA1A T VP F LILGNK IDIPYA A SED ELRY H LGLTN FTTGKG K VTL ATSARA1B E DSGVRPLEV FMCSIVRKMG YGEGFKWLSQ YI K ATSARA1A G DSGVRPLEV FMCSIVRKMG YGEGFKWLSQ YI N ATSARA1B ATSAR1B ATSAR1A Mass AA Sequence Mass AA Sequence 2335.198 1-19 MFLFDWFYGILASLGLWQK 2124.07 1-18 MFLFDWFYGILASLGLCK 292.3475 20-22 292.348 20-22 EAK EAK 1160.667 23-33 1160.667 23-33 ILFLGLDNAGK ILFLGLDNAGK 956.5597 34-41 TTLLHMLK 956.5597 34-41 TTLLHMLK 2129.099 45-63 LVQHQPTQHPTSEELSIGK 2129.099 45-63 LVQHQPTQHPTSEELSIGK 233.3232 64-65 IK 521.3082 64-67 INFK 257.3403 66-67 FK 1184.617 68-78 AFDLGGHQIAR 1184.617 68-78 AFDLGGHQIAR 659.3035 83-87 DYYAK 599.2494 83-87 DCYAK 1469.752 88-100 VDAVVYLVDAYDK 1497.758 88-100 VDAVVYLVDAYDR 581.2929 103-107 609.3242 103-107 FAESK FVESK 2342.285 109-130 ELDALLSDEALATVPFLILGN 2311.221 109-130 ELDALLSDEALANVPCLILG K NK 1491.733 131-143 1507.727 131-143 IDIPYAASEDELR IDIPYASSEDELR 1351.7 144-155 1377.705 144-155 YHLGLTNFTTGK YYLGLTNFTTGK 2433.263 156-177 GIVNLEDSGVRPLEVFMCSI 2178.141 158-177 VTLGDSGVRPLEVFMCSIVR VR 888.392 179-186 888.392 179-186 MGYGEGFK MGYGEGFK 923.4621 187-193 937.5142 187-193 WLSQYIN WLSQYIK 13

11/29/2012 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, etc. Sequencing with MS/MS • Currently three 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) – X!Tandem (hyperscore, the larger the better) 14

11/29/2012 Sequencing with MS/MS • 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 labeling 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

11/29/2012 SILAC ICAT-label • 4 parts to ICAT molecule – A protein reactive group – Iodoacetamide • covalently links to free cysteines . – An affinity tag – biotin • concentrates the cysteine-containing peptides, reducing complexity. 16

11/29/2012 ICAT-label • An isotopically labeled linker (C 10 H 17 N 3 O 3 ) – The linker chain can substitute up to nine 13 C atoms. – The light and heavy molecules are chemically identical – Comparison of labeled peptides provides a ratio of the protein concentration in the original sample. http://www.bio.davidson.edu/courses/GENOMICS/ICAT/ICAT.swf • ICAT-label • 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. 17

11/29/2012 iTRAQ • Label up to 8 samples at once • Amine specific labeling (lysine and N- terminal) (N-hydroxysuccinamide) • Mass of all labels the same. – The tag consists of the reactive group, a reporter molecule and a linker to balance the masses. – During fragmentation in MS the reporter group is released. • After fragmentation reporter labels are found between m/z 113 and m/z 121 • Ratio of peaks of reporter ions is proportional to relative concentrations. 18

11/29/2012 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 1-1000 ng) • DIGE 0.125 ng (linear 0.125 ng-10  g) • MS 500 fM • FTICR MS 500 aM – For a 60 kDa protein 500 fM = 30 ng 500 aM = 30 pg 19