Bioseparation Mass Spectrometry PDF

11/12/24 Mass spectrometry Dr. Rahul Modak...

11/12/24 Mass spectrometry Dr. Rahul Modak KIIT School of Biotechnology Course- Bio-separation (BT 4007) Reference: Handbooks of Agilent and Shimadzu 1 Introduction Mass spectrometry(MS) is an analytical chemistry technique that helps identify the amount and type of chemicals present in a sample by measuring the mass-to-charge ratio and abundance of gas-phase ions. A mass spectrum (plural spectra) is a plot of the ion signal as a function of the mass-to-charge ratio. From spectra, the mass of the molecular ion and fragments are used to determine the elemental composition or isotopic signature of a compound. This information is used to elucidate the chemical structures of molecules, such as pesticides or peptides. Mass spectrometry works by ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios. 2 1  11/12/24 Basic consideration Each element has specific mass Compounds, consisting of different elements, can be distinguished by their mass. Masses in Mass Spectrometry Average mass of a molecule is obtained by summing the average atomic masses of the constituent elements. Monoisotopic mass Accurate mass- H, O, N, C, S, Cl, Na, 3 Overview of MS Typical MS procedure: Sample (solid, liquid ,gas) is ionized Sample’s molecules might break into charged fragments during ionization Ions are separated according to their mass-to-charge ratio (m/z) Ions are detected by a mechanism capable of detecting charged particles (e.g. electron multiplier) Results are displayed as spectra of the relative abundance as a function of m/z ratio Identification is done by correlating known masses to the identified masses or through a characteristic fragmentation pattern 4 2  11/12/24 Modes of Ionization Gaseous Sample Introduction: Electron Ionization (EI) Chemical Ionization (CI) Liquid Sample Introduction: Electrospray Ionization (ESI) Atmospheric Pressure Chemical Ionization (APCI) Atmospheric Pressure Photo Ionization (APPI) Multimode Ionization (MMI) Matrix Assisted Laser Desorption Ionization (MALDI) Inductively Coupled Plasma (ICP) Polarity of analytes determines the ionization source. 5 Electron Ionization (EI) technique https://www.chem.pitt.edu/facilities/mass- spectrometry/mass-spectrometry-introduction 6 3  11/12/24 Chemical Ionization (CI) technique Positive ion mode: RH+ + A ------> AH+ + G Negative ion mode: [R-H]- + A ------> [A-H]- + G 7 Electrospray Ionization (ESI) technique https://www.youtube.com/watch?v=9AWBAI-Owzk&list=PL6yA4jv5tA-k9_2NVxm5jlzpZV_aW59DT 8 4  11/12/24 Atmospheric pressure chemical ionization (APCI) technique Multi-mode ionization: ESI+ APCI Ions from both ionization modes enter the capillary and are analyzed simultaneously by the mass spectrometer. Useful for screening of unknowns, or whenever samples contain a mixture of compounds where some respond by ESI and some respond by APCI. 10 Atmospheric solid Analysis probe ionization (ASAP) 11 5  11/12/24 Atmospheric pressure photo ionization (APPI) A+hv → A* A* → A+· + e- (IE < hv) If IE of analyte is more than photon (hv), A* may undergo photodissociation, photon emission. In such cases, use the substance called Dopant (D) which is promoting the ionization of analyte. D+hv → D+. D+. + A → D + A+· if EAA > EAD (Electron affinity) D+. + S → [D-H]· + [S+H]+ if PAS > PAD (Proton affinity) [S+H]+ → S + [A+H]+ if PAA > PAS 12 MALDI technique Matrix-assisted laser desorption/ionization (MALDI) is a technique to allows the high molecular weight compounds such as organic macro molecules and labile bimolecular into the gas phase as intact ions. MALDI is one of the recent developments of soft ionization techniques in the field of mass spectrometry. It can desorb intact analyte molecular ions with relative masses up to 300KDa. In MALDI-MS analysis, the analyte is first co-crystallized with a larger excess of a matrix compound (α-Cyano-4- hydroxycinnamic acid (CHCA), Dihydroxy benzoic acid (DBA), Sinapic acid etc, after which, on laser radiation of this matrix-analyte preparation results in desorption of the matrix as a plume, which carries the analyte along with it into gas phase. Thus the matrix plays a key role by strongly absorbing the laser light energy and causing, indirectly, the analyte to vaporize. The matrix also serves as a proton donor and acceptor, acting to ionize analyte in both positive and negative ionization modes, respectively. The TOF analyzers are typically used with the MALDI ionization source. 13 6  11/12/24 14 Mass Analyzer The mass spectrometer measures the ion signals resulting in a mass spectra, which can provide valuable information about the molecular weight, structure, identity, and quantity of a compound. Different types of mass analyzers: Single Quadrupole (SQ) Triple Quadrupole (QQQ) Time-of-Flight (TOF) Ion Trap (IT) 15 7  11/12/24 Quadrupole mass analyzer four parallel cylindrical metal electrodes with a hyperboloidal interior surface inside a vacuum chamber, positioned equidistant from the centre axis Both a direct current (D.C.) and high frequency alternating current or radiofrequency (RF) are applied. ions passing through this electric field oscillate in the x- and y- directions. When a given set of parameters are applied to the poles, certain ions of a specific m/z range maintain a stable oscillation. 16 Quadrapole principle: https://www.youtube.com/watch?v=6_mavZ_WKoU 17 8  11/12/24 A target ion with specific m/z is monitored. SIM on a single quad permits the best sensitivity for quantitation, however it lacks specificity. In Scan MS mode, the quadrupole mass analyzer is scanned sequentially allowing only 1 m/z at a time to pass to the detector. 18 Since the separating principle of the quadrupole MS systems is straightforward, they are comparatively easier to operate and maintain. Also, the quadrupole is compact in design, robust and relatively inexpensive. Consequently, they are widely adopted as a general-purpose analytical instrument. Furthermore, unlike other MS which require high vacuum levels, quadrupole MS can adequately function at lower vacuum levels (≒ 10-2 to 10-3 Pa). Even if they are interfaced with a GC or LC unit, the drop in vacuum level caused by the interface has minimal effect on the mass separation performance, making it the best suited for interfacing with chromatographic techniques. Quadrupole MS demonstrates good scan speed and sensitivity. With a maximum scan speed of 15,000 amu/second, it is capable of measuring at higher scan speeds than the magnetic sector MS. Its mass range can reach up to 2,000 m/z which enables the qualitative analysis in a practical range of molecular masses. In addition, it allows highspeed polarity switching, which facilitates simultaneous monitoring of multiple selected ions of different polarity. With the use of the SIM mode in a quadrupole MS, it can deliver a high-sensitivity quantitative analysis of a large number of target compounds, making it a widely recognized system among MS. 19 9  11/12/24 Mass Analyzer –Triple Quadrupole (QQQ) The analyzer consists of three quadrupoles (Q1-Q3) and therefore several modes of operation resulting in different information. A common set is the following: Q1: used as a filter for specific m/z(precursor ion) Q2: used as collision cell to fragment the precursor ion and generate product ions Q3: set to specific m/z(SRM or MRM) or scan mode (product ion scan) Information received: MS and MS/MS 20 Ion Trap (IT) MS Same principle as quadrupole MS. One donut shaped ring electrode and 2 end-capped electrode used. All the ions introduced and held in the trap together. Alteration of RF helps to IT-MS systems separate and detect masses by discharging ions inject one ion at a time. with unstable oscillations from the system. Information received: MS and MS/MS 21 10  11/12/24 As the name implies, ion trap MS systems trap the generated ions before separating them by mass. Consequently, they cannot perform selected ion monitoring (SIM) transitions and measurements for transmission type MS. Furthermore, they operate by pulsed mode and only a limited quantity of ions can be trapped, resulting in a narrower dynamic range than quadrupole MS systems. However, all trapped ions are detected in the IT MS, this provides higher sensitivity in scanning analysis than quadrupole models. In addition, it enables the trapping of a specific ion, fragmenting it and then trapping a specific product ion for further fragmentation, and so forth. Therefore, IT MS is considered a mass spectrometer specialized for elucidating the fragmentation pathway for structural determination of a target molecule. In addition to the two kinds of IT MS, there are other similar trapping type of MS such as the Fourier Transform Ion Cyclotron Resonance (FT-ICR) and Orbitrap. They adopt similar mechanism and principles. In the FT-ICR setup, the use of both the electric and magnetic field generates the stable oscillation and motion of the ions. To detect the ions, the selected ions are accelerated such that its radius of oscillating motion increases, the oscillation becomes unstable and eventually the ion gets removed. By determining the cyclotron frequency, it can be Fourier transformed and the ion mass is deduced. For Orbitrap MS systems, it only requires the use of electric field to trap and separate the ions. These MS systems demonstrate excellent mass resolution and mass accuracy. 22 Time-of-Flight (TOF) MS Time-of-Flight (TOF) MS is a pulsed and non-scanning MS. It has a simple construction, consisting of an accelerator, a field-free region, a reflectron and detector inside a high vacuum chamber called a flight tube. A high voltage pulse is applied which accelerates the ions into the flight tube. An ion mirror at the end of the tube reflects the ions and sends them to the detector that records their time of arrival. T: Time of flight m: mass of the ion v: velocity of the ion z: charge of the ion e: elementary charge V: acceleration voltage applied to ions L: flight distance in TOF 23 11  11/12/24 Mass analyzer Description Advantages Limitations Quadrupole Scanning Compact and simple Limited mass range Mass Filter Relatively cheap Low resolution Continuous Good selectivity (SIM) Little qualitative information Moderate vacuum required → well suited for coupling to LC Ion trap Trap Small and relatively cheap Limited dynamic range Pulsed High sensitivity Limited ion trap volume Good resolution Limited resolution Compact Requires pulse introduction to MS Time of flight Non-scanning High sensitivity and ion Requires pulsed introduction to (TOF) Pulsed transmission MS High resolution Requires fast data acquisition Excellent mass range Fast scan speed 24 Case studies- 1. Native protein mass detection 2. Protein identification through protease cleavage 3. Post translational modification determination 25 12  11/12/24 Mass spectrometers are now used for an extremely diverse range of applications, each with its own characteristics. MS system must be selected based on objectives, be it sensitivity, peak resolution, or a compact general-purpose system. In terms of cost and ease-of-operation, quadrupole mass analyzers have been increasing in market share for LCMS applications. Ion trap and TOF MS systems offer performance not obtainable from quadrupole models, so their usage and popularity is also continuing to increase. In summary, it is important to consider and choose a MS system that allows benefiting from the advantages offered by each ionization method and mass separation technique. MS facility at Univ. of Malborne, Aus. 26 13