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Mass Spectrometry Based Proteomics (1) Mass spectrometry based proteomics (1) Kenny Helsens [email protected] Department of Biochemistry, Ghent University Department of Medical Protein Research, VIB Ghent, Belgium Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 AMINO ACIDS AND PROTEINS Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Amino Acids and their properties From: http://courses.cm.utexas.edu/jrobertus/ch339k/overheads-1/ch5-amino-acids.jpg Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 A protein backbone H H O side chain R1 H O R1 R2 + N C C C O H C H O H C C H O + C N N C N O R2 O H O H H H H pepde bond amino group carboxyl group R1 H O R3 H O R5 H O R7 N N N O N H2N N N O R2 H O R4 H O R6 H OH amino terminus carboxyl residue terminus Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 A protein sequence R1 H O R3 H O R5 H O R7 N N N O N H2N 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 MGASYLR Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 MASS SPECTROMETRY: CONCEPTS AND COMPONENTS Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Schematic view of a generalized mass spec sample ion source mass analyzer(s) detector digizer Generalized mass spectrometer All mass analyzers operate on gas-phase ions using electromagne:c fields. Results are therefore plo?ed on a cartesian system with mass-over-charge (m/z) on the X-axis and ion intensity on the Y-axis. The laer can be in absolute or relave 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 digizer (ADC). Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Ion sources: MALDI laser irradiaon high vacuum h⋅ν + H + + + + + + + + + + + + + + + + + + + + + + + desorpon + + proton transfer + matrix Gas phase molecule analyte target surface Matrix Assisted Laser Desorp5on and Ioniza5on (MALDI) MALDI sources for proteomics typically rely on a pulsed nitrogen UV laser (υ = 337 nm) and produce singly charged pep:de ions. Compe::ve ionisaon occurs. The term ‘MALDI’ was coined by Karas and Hillenkamp (Anal. Chem., 1985) and Koichi Tanaka received the 2002 Nobel Prize in Chemistry for demonstrang MALDI ionizaon of biological macromolecules (Rapid Commun. Mass Spectrom., 1988) Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Ion sources: ESI www.sitemaker.umich.edu/mass-spectrometry/sample_preparaon m/z analyzer inlet + + + + + + + + + + + droplet evapora5on and + + charge-driven fission + + or ion expulsion + 3-5 kV + + + + + + + evapora5on only 0 + 0 0 0 0 N 0 0 0 2 0 0 0 0 sample 0 0 0 0 0 0 0 0 0 0 N2 0 needle nebulisaon Electospray ioniza5on (ESI) barrier ESI sources typically heat the needle to 40°to 100°to facilitate nebulisaon and evaporaon, and typically produce mul:ply charged pep:de ions (2+, 3+, 4+) John B. Fenn received the 2002 Nobel Prize in Chemistry for demonstrang ESI ionizaon of biological macromolecules (Science, 1989) – ESI is also used in fine control thrusters on satellites and interstellar probes… Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 ESI – online LC and solvents (nano) RP column (100–5 µm) (nano) needle spray solvent mixer aqueous solvent organic solvent H O + 0.1% FA 2 A B ACN + 0.1% FA + 2–5% ACN Nanospray ESI sources (5-10 µm diameter needle) achieve a higher sensi:vity, probably due to the higher surface-to-volume rao. For a spherical droplet this rao is: π ⋅r3 A = 4⋅π ⋅r 2 V = 4 3 A 3 6 = = V r D Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Analyzers: time-of-flight (TOF) high vacuum sample ions source detector field-free tube extrac:on (:me-of-flight tube) plate (30 kV) > 1 meter 2 m⋅v 2⋅ Ek Ek = q⋅V E = ↔ v = k 2 m We can now relate m/q (or the more commonly used m/z) 2 m 2⋅V 2⋅V 2⋅V ⋅t to the velocity of the ion, and using Newton’s kinemaca = 2 = 2 = 2 q v ⎛ x ⎞ x we can relate the speed to the travel :me and (known + ⎜ ⎟ exactly calibrated) field-free tube length ⎝ t ⎠ Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Analyzers: quadrupole (Q) + (U +V ⋅cosωt) permi?ed m/z ejected m/z − (U +V ⋅cosωt) 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. Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Analyzers: ion trap (IT) DC/ACRF voltage source detector capping ring capping electrode electrode electrode Ion traps operate by effec:vely trapping the ions in an oscillang electrical field. Mass separaon is achieved by tuning the oscillang 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. Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Fourier transform ion cyclotron resonance (FTICR) electrodes ion orbit strong magne:c field An FT-ICR is essen:ally a cyclotron, a type of par:cle accelerator in which electrons are captured in orbits by a very strong magne:c 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 superposi:on of sine waves is obtained. A Fourier transformaon is therefore required to tease out the individual ion frequencies. Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Fourier transform ion cyclotron resonance (FTICR) Movie h?p://medicine.yale.edu/keck/proteomics/technologies/mass_spectrometry/qicrvideo.aspx Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Orbitrap outer electrode inner electrode From: hTp://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 electrostac field in which the ions form an orbitally harmonic oscillaon along the axis of the field. The frequency of the oscillaon is inversely propor:onal 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. Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Resolution and why it matters Resolu:on in mass spectrometry is usually defined as the width of a peak at a given height (there is an alternave defini:on based on percent valley height). This width can be recorded at different heights, but is most oqen recorded at 50% peak height (FWHM). average monoisotopic mass mass From: Eidhammer, Flikka, Martens, Mikalsen – Wiley 2007 Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Detectors: electron multiplier single ion in 40V 20V 80V 60V 120V 100V 106 electrons out Different variaons of electron mul:plier (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. Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 TANDEM MASS SPECTROMETRY (TANDEM-MS, MS/MS, MS2) Kenny Helsens Mass spectrometry based proteomics (1) [email protected] EBI Bioinformatics Roadshow - Prague - 7 Sep 2011 Tandem-MS: the concept source detector ion selector fragment mass analyzer fragmentaon 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 func:on of ion selector, by selec:vely allowing only ions of a given m/z to pass through. The second mass analyzer is situated aer fragmentaon is triggered (see next slides) and is used in its normal capacity as a mass analyzer for the fragments.
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