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Paper 9: TECHNIQUES USED IN MOLECULAR BIOPHYSICS I

Module 26 : Principle of Mass Spectrometry

Dr.T.Velpandian, Professor

High Precision Bio-Analytical Laboratory, Ocualr Pharmacology and Pharmacy Division,

Dr.RPCentre, AIIMS, New Delhi

What is mass spectrometry?

“Mass spectrometry is the art of measuring atoms and molecules to determine their molecular weight”

1. Introduction

(Evolution of mass spectrometry, fundamentals, principle, construction)

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Mass spectrometer is a hyphenated instrument which measures the molecular weight of atoms and molecules after ionizing them. Often this instrument is coupled with chromatographic techniques like gas and liquid chromatography to handle complex matrices.

Together with separation property of chromatography, mass spectrometers render the precise identification and quantification of molecules and atoms from complex biosystems. For a chromatographer, tandem mass spectroscopy is a detector and for the mass analyst user, chromatography technique is a sample feeder. Although, it was originally developed for the identification of different atoms and their isotopes, o ver the time, several astonishing discoveries expanded its versatility in different fields of science viz, biology, chemistry, physics, medicine and space explorations.

2. Objective

This chapter explains you about the construction, working principle and application of mass spectroscopy with a special reference to quadrupole mass spectrometers in qualitative and quantitative analysis of small molecule in simple or complex mixtures.

- Evolution and development of mass spectrometry - Construction of mass spectrometer

- Types of mass spectrometers

- Basic concepts, fundamentals, principles and construction

History of Mass spectrometers

Sir J. J. Thomson of the Cavendish Laboratory of the University of Cambridge, discovered electron based on his experiments using electrical discharges in gases in 1897. The first mass spectrometer (parabola spectrograph) was developed for the determination of mass-to-charge ratios of ions. In this spectrograph, the influence of electric and magnetic fields were utilized for the ions move through parabolic trajectories using discharge tubes. Scintillation plate or photographic plates were used to record the deflection of ions. Subsequently, Thomson received the Nobel Prize in Physics in 1906. In Thomson's group, Francis W. Aston designed a mass spectrometer in which ions were dispersed by mass and focused by velocity. His model improved MS resolving power by an order of magnitude over the resolution better than the model developed by Thomson. Aston received the 1922 Nobel Prize in Chemistry for isotope studies carried out with this type of instrument. In 1920, Prof. A. J. Dempster, at Chicago developed a magnetic deflection instrument with direct ion focusing. He also developed the first electron impact source, which ionizes volatilized molecules with a beam of electrons from a hot wire filament.

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Noble prize winners in techniques associated with the current use of Mass Spectroscopy Ever since, several developments took place in the development of mass spectrometers for structural determination. In tandem MS (MS-MS), a precursor ion is mass-selected and typically fragmented by “collision-induced dissociation” (collisionally activated dissociation or CAD), followed by mass analysis of the resulting product ions. The collision- induced dissociation was introduced in 1968 by Prof. Keith R. Jennings, (Univ. of Warwick, England) and Prof. McLafferty (Univ. of Purdue).

Prof. R. Graham Cooks (Univ. of Purdue) defined the combination of newer soft ionization methods with collision- induced dissociation which gives tandem MS its power in the analysis of mixtures. The tandem MS instrument (triple quadrupole MS) was invented by Prof.

Richard A. Yost and Christie G. Enke (Michigan State Univ.). James D. Morrison of Latrobe University, Melbourne, Australia, helped Yost and Enke reduce the technique to practice.

Tandem MS was popularized by triple-stage quadrupoles introduced first by Finnigan and Sciex (in 1980).

Types of mass spectrometers

There are different types of mass spectrometers available for different applications. First question asked while looking for the help of mass spectrometer is what exactly the analyst wanted to know. The interest is only on molecular weight or in the quantity of particular molecule. Based on the requirement, the choice of machine varies. Looking forward for the precise estimation and analysis of compounds for their pure structural information one would opt for MALDI (Matrix Assisted Laser Desorption Ionization) coupled Time of Flight (TOF) or Quadrupole coupled Time of Flight. For pharmaceutical applications requiring quantification as well as metabolite identification, one would opt for either triple qudrupole or trap type of systems.

Instrument under review is liquid chromatography coupled triple quadrupole tandem mass spectroscopy.

Classical ‘U’ tube mass spectrometer

This is a basic instrument from where all other instruments got originated. Typically, this instrument contains a “U” tube which is kept under heavy vacuum. Next to the entry orifice the molecules entering in its vapor state meets a electron gun in which accelerated electron is allowed to impact on the molecules. This process ejects electron from the molecule thereby, the molecule in the gas state gains positive charge and becomes a molecular ion. This charged ion is accelerated to go through the tube to reach the detector to produce the signal. On its path, applied charge or magnetic field induces their deflection through the U tube. Using a

JJ Thompson Francis W Aston Wolfgang Paul Hans G Dehmelt John B. Fenn Koichi Tanaka

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right charge or magnetic field, molecule of interest focussing to the detector ultimately forms base peak in mass spectrum.

Figure showing the classical “U” tube mass spectrometer

Isotopic abundance of elements

Before understanding the principle and working of mass spectrometers, one must clear their idea about the fundamentals of elements. In the earth every element is existing along with their isotopes naturally. Isotopes have same atomic number with different molecular weights, for example hydrogen (MW=1) is having deuterium (MW=2) and tritium(MW=3), but all of them have atomic number as 1. In the standard mass spectrum, the isotopes form of compounds can be seen clearly. The mass of the most abundant peak in the mass spectrum is called mono-isotopic mass. Identifying the isotopic pattern is essential to determine the charge state of compounds in proteins which are known to have multiple charges and higher molecular weight. This aspect would be discussed in more detail in the further chapters discussing protein analysis using higher resolution mass spectroscopy instruments like Q- TOF.

Figure showing the isotopes of the compounds in the mass spectrum

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Types of mass spectrometers

During developmental stage, several methods were independently developed but today they are coupled together to handle wider applications in mass spectrometers. Basically, ions get deflected by both electric and magnetic fields, therefore, both of this techniques are having their application in the mass spectroscopy instruments. If only magnetic field is used, it fetches poor resolution, but together with electric field they gain importance for very high resolution. In a broad sense we can classify mass spectrometers as magne tic field deflection (alone or combined with electric field), quadrupoles (single, triple, ion-trap), time-of- flight (TOF), ion cyclotron resonance (FT-ICR) and others. Quadrupole type of instruments are the most popular type of hybrids, as they showed their typical function of storing and separating ions using alternating radio frequency and DC voltages. Therefore, the separating property of quadrupole is combined with electrical or magnetic deflectors to reach higher resolution in Q TOF type of instruments. With time the construction of mass spectrometers are widening to give place for accommodating newer applications.

General construction of mass spectrometer

This assembly consists of a sample feeder (HPLC/GC) coupled with an interface ionizer to a typical quadrupole mass spectrometer. In this section let us understand the individual components of quadrupole mass spectrometer.

Fig. 1.Sche matic representation triple quadrupole mass spectroscopy linked with a n ionize r to high performance liquid chro matography.

Parts of mass spectrometer (quadrupole type)

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In modern mass spectrometers, quadrupole type has been extensively used as a model for the separation of compounds due to their simple construction and speed of analysis. Several variants are available by different commercial manufactures and gained intellectual property rights for specific applications. As the name implies quadrupole consists of 4 cylindrical rods (looks like pencils) arranged in rectangular shape as shown in the schematic diagram.

These rods are supplied with alternating radio frequency and DC voltage diagonally to enable the isolation of ions according to their mass to charge ratio. These rods replaces the magnetic or charge mediated deflection in the classical ‘U’ tube mass spectrometer. When the qrudropoles are arranged in series, they are called tandem (repeats) to enable different mass spectroscopy experiments. This quadrupoles are kept in a stainless steel housing under higher dynamic vacuum using turbo molecular pumps to enable ion flying through vacuum as per the fundamental principle discovered by JJ Thompson. These quadrupoles are successfully used in both quantitative (triple quadrupole, Q Trap) and higher mass resolution in qualitative (Q-TOF) mass spectrometers. In either of the cases, separation of masses with a precision is expected to increase the speed of analysis when the chromatographically isolated compounds are continuously fed into the mass analysers.

It could be a genuine question of a student that why we need mass analyzer to do quad separation when it has already been separated chromatographically? To answer this question, in the 28th Chapter, the real working is demonstrated for fundamental understanding to answer this question. In brief, even after chromatographic separation, thousands of compounds could be seen in the mass analysers if the effluent is fed. There is a possibility of several compounds having lower molecular weights appearing simultaneously which is of botheration while analyzing a particular analyte with specific molecular weight. To enable the isolation of particular molecule weight, after its ionization, qruadrupoles are arranged in series to isolate, trap and fragment and to again select an ion which is a part of fragmented piece of the particular molecule selected. This kind of studies also gives understanding towards the structure details of the compounds for their elucidation.

Every mass spectroscopy instruments needs calibration using known molecular weight compounds. This is routinely done to tune and fix the radiofrequency with DC combination with m/z ratio. This calibration curve is applied through software in modern spectrometers for the mass analysis studies. In the classical hard ionization techniques like electron impact ionization the base peak is created by stripping an electron and making the molecule positively charged. Where as in the soft ionization techniques like Electro Spray Ionization (ESI) produces pseudo- molecular ions by forming hydrogen adducts (M+H)+ in the process.

Detailed information about this process is dealt in chapter 27 & 28.

Parts of mass analyser

If you look at a mass spectrometer from the sample entry side, the analyte isolated using chromatography techniques or sample infusion pumps enters into the ionizer compartment.

The ionizer compartment is again divided into two types based on the incidence of such ionization whether in atmospheric pressure or under vacuum. Soft ionization techniques like ESI happens at atmospheric pressure where as instruments like matrix assisted laser desorption ionization (MALDI) process and electron impact ionization process requires vacuum. The fundamental principle employed here is to convert sold phase substances (MALDI) or liquid phase substances into charged molecular ions which can be treated like

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the way electron is treated in the picture tube of the television to travel in the vacuum and are susceptible for attraction and repulsion for magnetic and electrical charges. It is not a problem for mass spectrometry as long as the molecule reaching into gaseous state from solid or liquid state either by sublimation or by evaporation. Once if the sample is not evaporated or present in liquid form/with matrix, the process of converting them into gaseous state for ionization to behave as a charged particle in vacuum is not feasible.

Therefore, soft ionization techniques like ESI are employed to enable substances like proteins, or eluents coming out of chromatographic techniques can continuously be handled in atmospheric pressure. Whereas in MALDI, laser (ultraviolet) evaporates the spot resulting in charge transfer from matrix to molecule in gas phase resulting in a molecule with a charge of +1.

Once the ionized compounds enter into mass analyzer either they are analysed using quadrupoles or using time-of- fight analysers of the purpose for which the mass spectrometery has been built.

Quantitative measurements in mass spectrometers

Typically, either single or triple quadrupole mass spectrometers are used for this purpose.

With the current development in technology one would expect the analysis of compound to the extent of famtomole is possible in this type of mass spectrometers. Therefore, these instruments are used in bio-analytical studies, screening for new-born metabolism errors, drug development, metabolism, targeted proteomic, lipodomic studies.

High accuracy mass determination (Qualitative) in mass spectrometers

This kind of instruments are used for the accurate determination of the molecular weight of proteins, peptides for their identification using the data bank like MASCOT, MassWizz, OMSSA, MyriMatch, SQID etc. After running 2D gel electrophoresis, usually newer bands are purified and subjected for mass determination analysis for their identification in the above databases for proteomic studies.

How to understand the differences in various types of mass spectrometers?

This question is valid for a student who is inclined to learn and use mass spectrometer for his/her studies. The mass spectrometers are broadly classified into aforesaid types, however, many hybrid instruments are developed to meet both extremes with considerable improvement in their respective read outs. The resolving power, mass accuracy, analytical sensitivity and the speed of analysis are the properties usually used to define their nature by default.

Mass Analyzer properties

Charge of a compound in soft ionization techniques

When a molecule is getting protonated (M+ H+)+ or deprotonated (M-H+)- arising from the soft ionization technique is expected to gain charge +1 or -1 respectively .

They follow the formula m+z

z (where m = Mass of the molecule and z = charge)

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According to this formula,

Singly protonated molecule with the molecular weight of 2000 would be seen in the mass spectra as

2000+1 = 2001 1

If the molecule is doubly protonated then, 2000 + 2 = 1001

2

If the molecule protonated 4 times at 4 various positions then 2000+4 = 501

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In electrospray ionization technique, a peptide having the molecular weight of 2000 would be reflected as having the molecular weight as 501. Here, the resolution power of the instrument is very important to understand the charge state.

Mass resolving powe r

The mass resolving power is the measure of its ability to distinguish two peaks of slightly different m/z. This property is of importance while determining accurate molecular weight of compounds for their identification from libraries. Usually the highest mass number of two adjacent peaks in the mass scan is used to derive this resolving power. Triple quadrupole mass spectrometers works with unit mass resolution but with higher sensitivity in quantification. Q-TOF type of instruments is known to have low sensitivity in quantification where as known to have higher mass resolution.

Mass resolution is calculated using the above formula where Mn = lowest mass number and Mm = highest mass number of two adjacent peaks. For example Mn=1999 and Mm=2000 then the resolution if 2000 by applying the above formula, which is seen in unit mass resolution mass spectrometers.

Other parameters used in mass spectrometry

The mass accuracy is the parameter which determine the accuracy of the instrument. It the ratio of the m/z measurement error to the true m/z. Based on the choice of mass analysers it could be varied ± 0.5 to ± 0.0001 m/z. Usually, ±0.5 is (nominal mass measurement) used for analytical purposes but for accurate mass ±5 ppm of the theoretical molecular weight is must.

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Mass range is the capability of the mass spectrometer in quantifying lower and higher values of analytes (molecular weight).

Linear dynamic range is the specification over which ion signal is linear with analyte concentration. Speed of the mass analyser refers to the time frames of experimental data taken per unit time. Speed governs the data points covered for quantitation experiments in the continuous monitoring mode where the effluent from sample injector is fed into mass analyser. According to the purpose of application, combination of various ionizing, separating and detection principles are employed in modern mass spectrometers.

References

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