BITS - Introduction to Mass Spec data generation

3,117 views
2,927 views

Published on

This is the first presentation of the BITS training on 'Mass spec data processing'.

It reviews the basic concepts of mass spectrometry data generation.

Thanks to the Compomics Lab of the VIB for contribution.

Published in: Technology
0 Comments
1 Like
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total views
3,117
On SlideShare
0
From Embeds
0
Number of Embeds
177
Actions
Shares
0
Downloads
95
Comments
0
Likes
1
Embeds 0
No embeds

No notes for slide

BITS - Introduction to Mass Spec data generation

  1. 1. http://www.bits.vib.be/training
  2. 2. mass spectrometry basics lennart martens lennart.martens@UGent.be Department of Biochemistry, Ghent University Department of Medical Protein Research, VIB Ghent, BelgiumLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  3. 3. AMINO ACIDS AND PROTEINSLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  4. 4. Amino Acids and their properties From: http://courses.cm.utexas.edu/jrobertus/ch339k/overheads-1/ch5-amino-acids.jpgLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  5. 5. A protein backbone H H O side chain R1 R2 + R1 H O C N C O H H C C O + H N C C O H N C C N O H R2 O O H H H H peptide bondamino group carboxyl group R1 H O R3 H O R5 H O R7 N N N O H2N N N N O R2 H O R4 H O R6 H OH amino terminus carboxyl residue terminusLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  6. 6. A protein sequence R1 H O R3 H O R5 H O R7 N N N O H2N N N N O R2 H O R4 H O R6 H OH Methionine Glycine Alanine Serine Tyrosine Leucine Arginine Met Gly Ala Ser Tyr Leu Arg M G A S Y L R MGASYLRLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  7. 7. MASS SPECTROMETRY: CONCEPTS AND COMPONENTSLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  8. 8. Schematic view of a generalized mass spec sam ple ion source m ass analyzer(s) detector digitizer Generalized mass spectrometer All mass analyzers operate on gas-phase ions using electromagnetic fields. Results are therefore plotted on a cartesian system with mass-over-charge (m/z) on the X-axis and ion intensity on the Y-axis. The latter can be in absolute or relative measurements. The ion source therefore makes sure that (part of) the sample molecules are ionized and brought into the gas phase. The detector is responsible for actually recording the presence of ions. Time-of-flight analyzers also require a digitizer (ADC).Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  9. 9. Ion sources: MALDI laser irradiation high vacuumh ⋅ν H+ + + + + + + + + + + desorption + + + + proton transfer + + + + + + + + + + + + matrix Gas phase molecule analyte target surface Matrix Assisted Laser Desorption and Ionization (MALDI) MALDI sources for proteomics typically rely on a pulsed nitrogen UV laser (υ = 337 nm) and produce singly charged peptide ions. Competitive ionisation occurs. The term ‘MALDI’ was coined by Karas and Hillenkamp (Anal. Chem., 1985) and Koichi Tanaka received the 2002 Nobel Prize in Chemistry for demonstrating MALDI ionization of biological macromolecules (Rapid Commun. Mass Spectrom., 1988)Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  10. 10. MALDI matrix molecules Matrix properties: • Small organic molecules • Co-crystallize with the analyte • Need to be soluble in solvents compatible with analyte • Absorb the laser wavelength • Cause co-desorption of the analyte upon laser irradiation • Promote analyte ionizationLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  11. 11. MALDI – spotting on target + matrix sample matrix + sample 1-2 mm spot on target and let air-dry stainless steel, gold teflon, … matrix crystalsLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  12. 12. Ion sources: ESI m/z analyzer inlet www.sitemaker.umich.edu/mass-spectrometry/sample_preparation + + + + + + + + droplet evaporation and + + + + + charge-driven fission + 3-5 kV + + or ion expulsion + + + + + + + evaporation only 0 N2 + 0 0 0 0 0 0 0 sam ple 0 0 0 0 0 N2 0 0 0 0 0 0 0 0 0 0 needle nebulisation Electospray ionization (ESI) barrier ESI sources typically heat the needle to 40°to 100°to facilitate nebulisation and evaporation, and typically produce multiply charged peptide ions (2+, 3+, 4+) John B. Fenn received the 2002 Nobel Prize in Chemistry for demonstrating ESI ionization of biological macromolecules (Science, 1989) – ESI is also used in fine control thrusters on satellites and interstellar probes…Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  13. 13. ESI – online LC and solvents (nano) RP column (100–5 µm) (nano) needle spray solvent mixer aqueous solvent organic solvent H2O + 0.1% FA A B ACN + 0.1% FA + 2–5% ACN Nanospray ESI sources (5-10 µm diameter needle) achieve a higher sensitivity,probably due to the higher surface-to-volume ratio. For a spherical droplet this ratio is: π ⋅ r3 A = 4 ⋅π ⋅ r 2 V =4 3 A 3 6 = = V r DLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  14. 14. Analyzers: time-of-flight (TOF) sample high vacuum ions source detector field-free tube extraction (time-of-flight tube) plate (30 kV) > 1 meter m ⋅ v2 2 ⋅ Ek Ek = q ⋅ V Ek = ↔v= 2 m We can now relate m/q (or the more commonly used m 2 ⋅V 2 ⋅V 2 ⋅V ⋅ t 2 m/z) to the velocity of the ion, and using Newton’s = 2 = 2 = q v  x x2 kinematica we can relate the speed to the travel time   and (known + exactly calibrated) field-free tube length tLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  15. 15. Analyzers: time-of-flight – reflectron Ekin > Ekin ion mirror reflectron detector with reflectronLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  16. 16. Analyzers: time-of-flight: delayed extraction source extraction plate (0 kV) source extraction plate (0 kV) **** *** ** * field gradient source extraction plate (30 kV) time t0 tx t x + 100 nsLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  17. 17. Resolution and why it matters Resolution in mass spectrometry is usually defined as the width of a peak at a given height (there is an alternative definition based on percent valley height). This width can be recorded at different heights, but is most often recorded at 50% peak height (FWHM). average monoisotopic mass mass From: Eidhammer, Flikka, Martens, Mikalsen – Wiley 2007Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  18. 18. Analyzers: ion trap (IT) DC/ACRF voltage source detector capping ring capping electrode electrode electrode Ion traps operate by effectively trapping the ions in an oscillating electrical field. Mass separation is achieved by tuning the oscillating fields to eject only ions of a specific mass. Big advantages are the ‘archiving’ during the analysis, allowing MSn. Wolfgang Paul and Hans Georg Dehmelt received the 1989 Nobel Prize in Physics for the development of the ion trap.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  19. 19. Analyzers: quadrupole (Q) + (U + V ⋅ cos ωt ) permitted m/z ejected m/z − (U + V ⋅ cos ωt ) ejected m/z Quadrupole mass analyzers also use a combined RF AC and DC current. They thus create a high-pass mass filter between the first two rods, and a low-pass mass filter between the other two rods. The net result is a filter that can be fine-tuned to overlap(and thus permit) only in a specific m/z interval; ions of all other m/z values will be ejected.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  20. 20. Detectors: electron multiplier single ion in 40V 20V 80V 60V 120V 100V 10 6 electrons out Different variations of electron multiplier (EM) detectors are in use, and they are the most common type of detector. An EM relies on several Faraday cup dynodes with increasing charges to produce an electron cascade based on a few incident ions.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  21. 21. Fourier transform ion cyclotron resonance (FTICR) electrodes ion orbit strong magnetic field An FT-ICR is essentially a cyclotron, a type of particle accelerator in which electrons are captured in orbits by a very strong magnetic field, while being accelerated by an applied voltage. The cyclotron frequency is then related to the m/z. Since many ions are detected simultaneously, a complex superposition of sine waves is obtained. A Fourier transformation is therefore required to tease out the individual ion frequencies.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  22. 22. Orbitrap From: http://www.univ-lille1.fr/master-proteomique/proteowiki/index.php/Orbitrappe An OrbiTrap is a special type of trap that consists of an outer and inner coaxial electrode,which generate an electrostatic field in which the ions form an orbitally harmonic oscillation along the axis of the field. The frequency of the oscillation is inversely proportional to the m/z, and can again be calculated by Fourier transform. The OrbiTrap delivers near-FT-ICR performance, but is cheaper, much more robust, and much simpler in maintenance. It is a recent design, only a few years old.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  23. 23. TANDEM MASS SPECTROMETRY (TANDEM-MS, MS/MS, MS2)Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  24. 24. Tandem-MS: the concept source detector ion selector fragm ent m ass analyzer fragm entation Tandem-MS is accomplished by using two mass analyzers in series (tandem) (note that a single ion trap can also perform tandem-MS). The first mass analyzer performs the function of ion selector, by selectively allowing only ions of a given m/z to pass through. The second mass analyzer is situated after fragmentation is triggered (see next slides) and is used in its normal capacity as a mass analyzer for the fragments.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  25. 25. Why tandem-MS? peptide structure x3 y3 z3 x2 y2 z2 x1 y1 z1 R2 R3 R1 CH2 CH2 R4 NH2 C CO N C CO N C CO N C COOH H H H H H H H a1 b1 c1 a2 b2 c2 a3 b3 c3 There are several other ion types that can be annotated, as well as ‘internal fragments’. The latter are fragments that no longer contain an intact terminus. These are harder to use for ‘ladder sequencing’, but can still be interpreted. This nomenclature was coined by Roepstorff and Fohlmann (Biomed. Mass Spec., 1984) and Klaus Biemann (Biomed. Environ. Mass Spec., 1988) and is commonly referred to as ‘Biemann nomenclature’. Note the link with the Roman alphabet.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  26. 26. Creating fragments (unimolecular) laser (( )) activated ion precursor ion (metastable) non-activated ion fragment ions (stable) 200 400 600 800 1000 1200 1400 1600 m/z This fragmentation method is called post-source decay (PSD) and relies on a single unimolecular event, in which a highly energetic (metastable) ion spontaneously fragments. PSD typically causes backbone fragmentation. y and b ions are by far the most prevalent fragment types, although TOF-TOF instruments (see later) specifically also yield substantial internal fragments.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  27. 27. Creating fragments (bimolecular I) gas inlet collision cell collision gas (atom or molecule) selected peptide ∆V This fragmentation method is called collision-induced dissociation (CID) and relies on a series of bimolecular events (collisions) to provide the peptide precursor with sufficient energy to fragment. CID typically causes backbone fragmentation. y and b ions are by far the most prevalent fragment types. The collision gas is typically an inert noble gas (e.g.: Ar, He, Xe), or N2.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  28. 28. Creating fragments (bimolecular II) electron source - fragm entation cell electron - - - - - selected peptide ∆V This fragmentation method is called electron-capture dissociation (ECD) or electron- transfer dissociation (ETD) and relies on a single impact of an electron on a peptide precursor. This high-speed impact immediately imparts sufficient energy to fragment the precursor (non-ergodic process). Like CID, ETD and ECD typically cause backbone fragmentation, but they typically result in c and z ions. ECD is only workable in FT-ICR mass spectrometers, whereas ETD is used in traps.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  29. 29. A CID FRAGMENTATION PRIMERLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  30. 30. CID fragmentation: the mobile proton model H+ Assum ption peptide H+ Hypothesis peptide X fragmentation event 14 pK a values 12 R–group 10 NH2– pKa 8 6 4 –COOH 2 0 H ine Ty an e ne Ph thio e id Pr e e Ar n e yl ne ne Le e re e yp ne e G e e C cid rti e e ta tein n in n rin lin in Is din in in pa gin Ac ph si si i en ni As ni ci Tr oni A c an an ol m uc Va Se ly ro Ly eu gi ti As ara ys ta to ic c is al Al m ol lu Th p e G M lu GLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  31. 31. Illustration of the MPM hypothesis The necessary collision energy to achieve a given fragmentation efficiency is highest and equal for both 1+ peptides on the left (the R present in both sequesters the charge), and is higher for PPGFSPFR for the 2+ peptides. This is because the latter has an N-terminal P, which has a relatively high pKa for the N-terminus; so the second available charge is located at either end of the peptide. The other peptide has no real main contender (in pKa) for the second charge, and therefore fragments more easily. On the right, having two R’s in the sequence negates the effect of double charge (both charges are sequestered by an R). From: Wysocki et al, J. Mass Spectrom., 2000Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  32. 32. Ionization and fragmentation peptide 1+ fragmentation b-ion y-ion 1+ ? peptide 2+ fragmentation 1+ 1+ b-ion y-ionLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  33. 33. A SPECIAL FLAVOUR OF MS/MS: MULTIPLE REACTION MONITORINGLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  34. 34. Multiple Reaction Monitoring (MRM) mass filter 1 collision mass filter 2 cell peptide selected fragments selected mixture peptides of both fragment peptides MRM removes noise, yielding better signal-to-noise ratio MRM removes ‘contaminating’ peaks, aiding targeted identification MRM works well with proteotypic peptides MRM can be performed with Q-Q-Q, Q-LIT and IT instrumentsLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  35. 35. MRM analysis less complex MS1 analysis peptide fractions MS2 analysis fragment selection Multiple Reaction Monitoring is the most often used name for this method, even though the IUPAC draft standard for mass spectrometry does not endorse this term, and prefers Single Reaction Monitoring (SRM) instead. You can find both terms in use.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  36. 36. MASS SPECTROMETER CONFIGURATIONSLennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  37. 37. ESI ion trap ESI detector source ion trap Very simple and reliable instrument, that can perform MS and CID MS/MS thanks to the ion trap. Mass accuracy is relatively poor however, and the resolution is lacking as well (unable to distinguish isotopes, hampering charge state determination).Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  38. 38. ESI triple quadrupole ESI detector source quadrupole 1 quadrupole 2 quadrupole 3 Simple instrument, that recently attracted attention because it is well-suited for Multiple Reaction Monitoring (MRM). It can perform MS and MS/MS, where the first quadrupole is the ion selector, the second quadrupole a collision cell and the third quadrupole a mass analyzer. Due to the ‘wastefulness’ of a quadrupole as mass analyzer, it is not very popular for general MS/MS analysis, however.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  39. 39. MALDI DE RE-TOF reflectron detector M ALDI linear source TOF tube detector delayed reflectron ex traction voltage gate for precursor selection Relatively simple instrument, that can measure intact masses quite accurately and can perform fragmentation analysis through PSD (unimolecular fragmentation), but the yield of fragmentation is poor. Cheap and reliable, but out of date nowadays.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  40. 40. MALDI TOF-TOF reflectron detector M ALDI source 2 linear source TOF 1 TOF 2 detector delayed reflectron ex traction voltage gate for precursor selection A modern version of the MALDI DE RE-TOF, the TOF-TOF relies on two TOF tubes in tandem. The second TOF is fitted with a reflectron. Mass accuracy and resolution are very high and the instrument can perform MS and MS/MS, both for peptides as well aswhole proteins. The archiving nature of the MALDI targets allows the instruments to scana single sample more thoroughly. TOF-TOF instruments are also well-suited for profiling.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  41. 41. ESI quadrupole time-of-flight (Q-TOF) pusher detector ESI source quadrupole TOF collision cell reflectronOne of the first hybrid mass spectrometers, the Q-TOF combines the excellent precursorselection characteristics of the quadrupole with the mass resolution and accuracy of the TOF tube. The ESI source produces multiply charged ions, and can be coupled to anonline (nano-) LC separation. A Q-TOF is a very good instrument when high-quality dataare more important than high-throughput, and is well suited for protein characterisation.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  42. 42. ESI quadrupole linear ion trap source detector capping quadrupole capping electrode electrode Linear ion traps store ions in a fixed linear trajectory rather than in a ‘blob’. The major advantage is the increased capacity for trapping ions. This allows for a broader dynamic range and better quantitation performance. The linear ion trap isthese days often encountered as the workhorse mass spectrometer in many labs, andcan be extended with the highly accurate orbitrap or FT-ICR mass analyzers/detectors.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  43. 43. ESI linear ion trap FT-ICR or Orbitrap C-trap ESI linear ion trap source FT-I CR Orbitrap The combination of a linear ion trap and a high-resolution, high-accuracy FT analyzer allows for a broad dynamic range and highly accurate mass measurements. Since the ion trap can be used as a collision cell, the FT analyzer can also measure the resulting fragments with high accuracy.Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011
  44. 44. Thank you! Questions?Lennart Martens BITS MS Data Processing – Mass Spectrometrylennart.m artens@UGent.be Gent, Belgium, 16 December 2011

×