High Throughput Protein Analysis Enabled by

IR, MALDI, ESI, MS

Pathan Najiya Shahnoor*, Sayyad Simran Shabbir, Shaikh Sadiya Mehmood, Ansari Shaheenanjum Babuddin, Dr. Aejaz Ahmed,  Dr. G. J.  Khan.

J.I.I.U' S Ali Allana College of Pharmacy Akkalkuwa, Dist. Nandurbar (425415), Maharashtra, India

Abstract: With the introduction of soft ionization techniques such as Matrix Assisted Laser Desorption Ionization (MALDI), and Electrospray Ionization (ESI), proteins have become accessible for mass spectrometric analyses. Since then, mass spectrometry has emerged as the preferred technique for identifying proteins and peptides at a low cost, with high reliability and sensitivity. With a growing number of complete genome sequences available for a wide range of organisms and multiple protein databases built from them, mass spectrometry can now identify proteins with high throughput. This Review discusses the suitability of strategies for automated data analysis and provides a brief overview of methods for identifying posttranslational modifications, highlighting the various mass spectrometric techniques currently used in proteome research. In the biopharmaceutical sector, mass spectrometry (MS) is the main analytical method used to identify proteins. The gold standard method for analysing intact proteins is to combine liquid chromatography with electrospray ionisation (ESI). Nevertheless, speed constraints make it impossible to analyse big sample sizes (>1000) in a single day. On a high throughput IR-MALDESI-MS system, proteins as small as 150 kDa can be detected, and we have assessed the impact of the matrix on the signal. Up to 22 protein samples can be analysed by the system in a single second. Applications include compound modifications to a probe protein, compound binding kinetics, and protein autophosphorylation. A cysteine modification site was found using top-down protein sequencing. Keywords:. Mass spectrometry, ESI (Electrospray Ionization), MALDI (Matrix Assisted Laser Desorption Ionization)

Corresponding Author: Pathan Najiya Shahnoor   Email ID: najiyasp@gmail.com

Article History Received:        15/09/2023 Revised:          02/10/2023 Accepted:        24/10/2023 Published:       06/11/2023

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

INTRODUCTION

Mass spectrometry (MS) is a widely used technique for analysing chemical structures in quantities down to trace levels. It was first developed in 1905. Proteins were unavailable for mass spectrometry analysis for many years due to inadequate ionisation methods for large biomolecules. Since the advent of soft ionisation methods like Electrospray Ionisation (ESI) and Matrix Assisted Laser Desorption Ionisation (MALDI) at the close of the 1980s, protein analysis by mass spectrometry has rapidly advanced. Simultaneously, a growing number of complete genome sequences for diverse organisms have become accessible, and multiple protein databases have been built using this data. High-quality, well-annotated protein databases provided the foundation for high-throughput protein identification using mass spectrometry. [12]. Many different types of mass spectrometric instrumentation, such as MALDI-TOF, ESI-Q-TOF, ESI-ion trap, MALDI-TOF/TOF, ESI-FT-ICR, etc., have been created by combining different types of mass analyzers in a modular arrangement with MALDI- or ESI. While the primary structure of a protein could be determined using each of these MS techniques, additional sample preparation methods were always needed. Moreover, it is now feasible to analyse posttranslational modifications like glycosylation and phosphorylation. High sensitivity, mass accuracy, mass resolution, fast analysis, and sophisticated data handling are now combined in system-dependent mass spectrometers.In addition to these technical aspects in mass spectrometry, greatly improved sample separation and preparation techniques have also led to enhanced sensitivity. The quantification of chemically or metabolically labeled proteins is yet another focus of interest in mass spectrometry. Despite these advances, current MS approaches still have limitations and are therefore subject to further development. Thus, the purpose of this paper is to provide an overview of the various mass spectrometric techniques currently used in proteome research, as well as to discuss the suitability of these techniques for protein quantification strategies. The methods for identifying post-translational modifications are briefly discussed. [13].

General technical considerations:

A very precise and sensitive technique for figuring out the molecular masses of various kinds of molecules is mass spectrometry. Every typical mass spectrometer is made up of three main parts:

Ø  The ion source ionizes the analyte.

Ø  The mass analyzer separates the resulting ions according to their mass-to-charge ratio (m/z).

Ø  The ion detector, whose signals can be recorded and processed by a computer.

Fig.1: MALDI TOF Mass Spectrometer

This order indicates the direction of the ion's passage through a typical mass spectrometer.13] There are various designs available for each mass spectrometer unit, and they can all be set up in a variety of ways. For mass spectrometers in Specifically, various unit configurations can be integrated into a single mass.The spectrometer. For example, the coupling of two mass selective devices for tandem mass spectrometry (MS/MS) has expanded the field’s application enormously, resulting in a profusion of experimental setups and designs in modern protein analyzing mass spectrometers. For a better understanding of the variety of instrumentation, a brief introduction to the functional principles of the most common designs is essential.

Ion sources:

The purpose of the ion source is to produce analyte ions and move them into the gas phase so they can enter the mass spectrometer's vacuum. Loss or gain of charge (such as electron capture, electron ejection, protonation, deprotonation, or cationization) produces the ions. Prior to the development of MALDI and ESI ionisation, electron ionisation (EI) was the most widely used ionisation technique for mass analysis. Due to the volatility of large biomolecules in a vacuum by thermal desorption, the electron ionisation technology is limited to compounds with masses well below the range of peptides and proteins. Nevertheless, routine analysis of small molecules still heavily relies on electron ionisation.

The First Protein identification using mass spectrometry:

Although there are still a number of restrictions, methods like fast atom bombardment (FAB) and plasma desorption have been successful in producing a biomolecule ionisation that is satisfactory. The advent of "soft" ionisation techniques in mass spectrometry, such as MALDI and ESI, has the potential to address issues related to thermal decomposition and excessive fragmentation of large biomolecules, like peptides. In both situations, the analyte is firstly protonated in a liquid phase and then augmented with a proton donor (such as an organic acid) to achieve ionisation.

 ESI: (Electrospray ionization)

Using varying amounts of an aqueous sample at atmospheric pressure, charged molecules are introduced into the mass spectrometer equipped with ESI sources. For instance, in nano electrospray (nanoES) technology, a metal-coated glass needle's highly charged tip (up to 3,000 V) can be sprayed with a small amount of sample all the way to the mass spectrometer's inlet (Fig. 2). Strong electric field produced by the finely pointed nozzle helps to accelerate charged droplets and create a continuous spray of 20–200 nL/min. After the solvent evaporates, which is often assisted by a dry gas, the solvent-free ionised analyte molecules are eventually released by decreasing the droplet size and raising the surface charge density. In this case, organic solvents like acetonitrile or 2-propanol help to create a stable spray by speeding up the evaporation process. To create an ion beam, the resultant ions are guided through an aperture and electrostatically focused step-by-step under increasing vacuum conditions. The principal product of the ESI process is multiply charged molecules. It is shown that solvents more volatile than water affect the maximum charge states and charge state distributions of ions produced by electrospray ionisation.

 

MALDI (Matrix Assisted Laser desorption/Ionization):

Using the ionisation technique known as MALDI spectrometry, one can accurately determine the molecular mass of polar biopolymers with molecular masses ranging from a few thousand to several hundred thousand Da. In 1988, the technique was initially presented almost simultaneously by two research teams—one from Germany and the other from Japan. For MALDI, commercial instrumentation is available. The MALDI technique involves uniformly dispersing a low concentration of analyte in a solid or liquid matrix and depositing it on a metal plate or the end of a stainless steel probe. After that, the plate is put inside a vacuum chamber, and the sample is targeted by a laser beam [5]. Apart from the standard MALDI conducted in a vacuum chamber, an atmospheric version has also been reported. The laser radiation must be thoroughly absorbed by the MALDI Matrix. An ion plume is then produced by desorbed and ionising the matrix and analyte. The time of flight (TOF) analyzer is the most popular kind of mass analyzer used with MALDI. a mass spectrum obtained using a MALDI-TOF device. In this instance, the analyte was a monoclonal antibody from a mouse with a molecular mass of roughly 150,000 Da, and the matrix material was nicotinic acid.

)

Fig.2 Electrospray Ionization (ESI

Note that the Spectrum is characterized by very low background noise and a complete absence of fragmentation of the large analyte ion. Multiply charged ions are present as well as peaks for dimer and trimer species. Though the exact mechanism underlying the formation of the MALDI ion plume is unknown, it is believed to entail the Matrix absorbing the laser beam and then transferring that energy to the analyte. The analyte and the matrix then undergo desorption.The analyte is thought to desorb as natural molecules and then to be ionized by proton-transfer reactions with a protonated matrix. A series of photochemical reactions may produce protonated matrix ions. Along with the laser wavelengths that have been used. Lasers used include Nitrogen (337 nm) Nd-YAG (266 and 355 nm), excimer (308 nm), Er-YAG(2.94µm), and CO² (10.6µm). The most common Sample preparation method is the dried-droplet technique in which the droplet of the Matrix containing the analyte is deposited on the metal plate and then dried. Typically, the ratio of analyte to Matrix is 1:10³ to 1:10⁵. Analyte concentrations are usually in the micromolar range. Recently, The MALDI Technique has been extended to imaging methods by scanning a localized laser beam over the dispersed sample. A mass spectral variety of biopolymers has been obtained in this manner.

 

1.      Mass analyzers:

          i.            Time of Flight (TOF):

Mass analyzer an attractive feature of the TOF mass spectrometer is its graspable design. The mass analysis simply involves measuring the flight time of the ions on their way through the field-free-drift region in a flight tube after acceleration. The m/z values of the ions in the analyzer tube determine their velocity.The greater the m/z, the lower the speed and the longer the time needed to travel the distance to the detector [3]. Unfortunately, for a simple linear tube design, the mass resolution is relatively poor due to the inevitable initial energy spread from the evaporation process.

 

Fig.3: Matrix-Assisted Laser Desorption/Ionization (MALDI TOF)

The reflectron, which is at the end of the flight tube and compensates for fuzziness in flight times by focusing ions with the same m/z in space and time before they hit the detector, was introduced to eliminate this disadvantage (Fig. 3). Therefore, it is easy to achieve high resolution up to 25,000 with a reflectron TOF mass analyzer design. The post-source decay (PSD) technique, which makes use of the fact that some of the MALDI-generated ions experience metastable decay during flight through the mass analyzer, is another feature of MALDI-TOF instruments.For simple reflectron MALDI-TOF devices, a composite PSD mass spectrum is generated stepwise due to the kinetic energy range-dependent focusing potential. However, modern MALDI TOF-TOF-MS devices provide a faster and more precise MS/MS-spectrum generation comparable with other common tandem-MS devices [3].

 

 

        ii.            Quadrupole (Q or Quad) mass analyser:

The m/z-dependent trajectory of ions in an alternating radio frequency field forms the basis of the quadrupole mass filter concept. Two pairs of rod electrodes that focus ions in two dimensions—that is, two axes—create the oscillating field. To reach the active attracting electrode, the ions are accelerated alternately. A portion of the ions with a particular m/z value are stabilised in between the electrodes at any given field oscillation of the amplitude and frequency, while the majority of ions are discarded. Quadrupole mass analyzers are therefore referred to as mass filters. Ions can either drift through a third dimension as in quadrupole mass filters or be trapped in an ion trap with various electrode designs. The scanning mass gate's range is heavily dependent on field modulation. An increase in the mass window causes more of the selected ions to travel through the analyzer on stable trajectories, which boosts signal but lowers resolution. Tandem MS with quadrupole mass analyzers is frequently performed using the triple quadrupole (triple quad) and Q-TOF mass spectrometer setups.

Fig.4 Mass Analysers

Ø  TOF: Certain time-of-flight reflectron analyzers offer high mass resolution and can perform LIFT- or PSD-tandem MS.

Ø  Ion Trap: Although the Paul ion trap typically has poor mass resolution and accuracy, it can conduct MSn experiments quickly.

Ø  Quad: Tandem mass spectrometry with high mass accuracy can be accomplished with a collision cell and multiple quadrupole mass filters.

Ø  LIT: A combination of an ion trap analyzer (connecting arrows) and a quadruple simplifies linear ion traps while improving overall performance. It can hold ions in strings within the end caps.

Ø  Orbitrap: It can be regarded as an extremely well-resolved and accurately mass-measured modified ion trap.

      iii.            FT-ICR: Of all the devices that are currently on the market, this mass analyzer offers the best mass accuracy and the highest resolution power. There are numerous ways to combine these analyzers with ion sources, detectors, and each other.

      iv.            Ion trap mass analyzer:

 The ion trap functions similarly to a quadrupole analyzer in theory; the only distinction is that the electrode assembly of the ion trap traps the ions in three dimensions. A compact ring electrode and two end-cap electrodes define the trapping volume for specific ions. Ion trap analyzers operate at a higher level of sophistication because they can perform complex mass analyses using multiple gate drives. A triple quadrupole mass spectrometer and an ion trap device operate similarly in many aspects. The ion trap conducts each operation sequentially in a single device that is only separated in time, whereas the triple quad performs ion selection, collisional dissociation, and mass analysis in three aligned mass analyzers that are separated in both time and space. The cap on the quantity of ions that can be trapped is a significant flaw in the ion trap design. More ions can be seen to deviate from their expected behaviour and interact with one another more—for example, by repelling one another when their charges are identical—the more ions are contained within the ion trap's small volume. Excessively high ion density directly results in a significant loss of mass accuracy and resolution. To guarantee that an appropriate quantity of ions is captured during each scan, extra scanning and control methods are needed to account for this "space charge" phenomenon. Usually, 0.5 amu can be fixed with ease if "space charging" is reduced. An MS/MS spectrum can be produced by scanning the fragment ions out of the trap after collision-induced dissociation (CID). Ion trap instruments (MSn) can typically be used to perform additional MS stages if necessary. However, depending on the ion yields from earlier experiments, n is typically less than 7. Ion trap mass analyzers have many benefits, including robustness, flexibility, sensitivity, fast scanning rates, and relatively low costs.

Orbitrap:

 The Orbitrap can be thought of as a highly modified ion trap, even though it uses constant electrostatic fields instead of the oscillating electric field used in the ion trap (Fig. 4). The Orbitrap's electrode geometry is an entirely new design that has an electrode in the centre that resembles a spindle and an extended circular outer barrel. The combined quadro-logarithmic electro-static potential produced by these axially symmetric electrodes results in stable ion trajectories around the central electrode and concurrent axial oscillation. The Orbitrap design can be used with both MALDI and ESI sources and offers a high dynamic range, high mass accuracy (2-4 ppm), and high resolution (up to 150,000). While various laboratories are currently examining the Orbitrap's suitability for tandem mass spectrometry, this new breed of high-resolution mass analyzer has the potential to become a more affordable option than FT-ICR-MS equipment. Unfortunately, there are currently not enough real-world data to assess how Orbitrap instruments will affect mass spectrometric protein analysis in the future.

        v.            Fourier Transform-Ion Cyclotron Resonance (FT-ICR) mass analyzer:

 The analysis of proteins and peptides derived from MS samples is greatly impacted by this intelligent kind of mass analyzer. Compared to other mass spectrometer designs that are presently on the market, FT-ICR-MS provides a higher mass accuracy and resolution. The high performance of the FT-ICR analyzer is a result of the analyte ions being trapped in a mixture of strong magnetic and electric fields (Fig. 4). In the presence of a uniform static magnetic field, ions trapped by a static electric field are confined to circular orbits. The m/z of the ion and the strength of the magnetic field determine the frequency of the circular motion, or cyclotron frequency. The momentum of the ions in the plane perpendicular to the magnetic field determines the radius of this circular motion. Ions can therefore be contained for extended periods of time in high vacuum, and cyclotron frequency detection and ion excitation can be repeated. This method enables the simultaneous, non-destructive acquisition of all ions' spectra using a broadband amplifier after the ions have been detected. A complete mass spectrum with extremely high mass accuracy can be obtained by Fourier transformation of the induced image current signals. Regretfully, FT-ICR-MS performance improves in all respects at higher magnetic fields, which are typically generated by superconducting magnets. Presently accessible superconducting magnetic materials usually require operation at very low temperatures.

3. Ion detectors:

 Destructive ion detection is the standard method for registering incoming ions from the various mass Protein identification using mass spectrometry, with the exception of the Orbitrap and the FT-ICR instruments: An analysis technique. Microchannel plate (MCP) detectors or secondary electron multipliers (SEM) are commonly used to detect ions. Typically, the detector produces secondary electrons that are amplified further, allowing the mass spectrometer to produce an analogue signal. A computer finally digitises and processes the analogue signal from the detector. There are a number of other ion detection designs and applications in use, such as photon-sensitive detectors, but they are outside the purview of this review.

2.      Instrumentation of MALDI-MS:

The MALDI technology has multiple variants, and similar instruments are now manufactured for radically different uses. From higher throughput and more industrial to more academic and analytical. The field of mass spectrometry has grown to the point where more high-throughput instruments and ultrahigh resolution mass spectrometry, like the FT-ICR instruments, are needed. Since a variety of interchangeable ionisation sources (electrospray, MALDI, atmospheric pressure, etc.) are available for purchase with many MALDI

 

MS instruments, there is often overlap between the technologies and the potential to use any soft ionisation technique.

Fig.5 MALDI MS Components

i.                    Laser:

UV lasers such as nitrogen lasers (337 nm) and frequency-tripled and quadrupled Nd:YAG lasers (355 nm and 266 nm, respectively) are commonly used in MALDI techniques.

The mid-IR optical parametric oscillator, the 10.6 μm carbon dioxide laser, and the 2.94 μm Er: YAG laser are examples of infrared laser wavelengths used for infrared MALDI. Because of their softer mode of ionisation, infrared lasers are used, despite being less common. More material removal (good for biological samples), reduced low-mass interference, and interoperability with other matrix-free laser desorption mass spectrometry techniques are further benefits of IR-MALDI.

ii.                  Time of flight:

The time-of-flight mass spectrometer (TOF) is the most popular mass spectrometer type when used with MALDI, primarily because of its wide mass range. Since the pulsed laser fires in discrete "shots" as opposed to continuously, the TOF measurement technique is also perfectly adapted to the MALDI ionisation process. A reflectron, also known as a "ion mirror," is frequently included with MALDI-TOF instruments. It uses an electric field to reflect ions. By lengthening the ion flight path, this improves resolution by lengthening the time between ions with different m/z [4].

Resolving power m/Δm of contemporary commercial reflectron TOF instruments can attain 50,000 FWHM (full-width half-maximum) or higher. Δm is defined as the peak width at 50% of peak height.

To distinguish between phosphorylated and non-phosphorylated peptides, MALDI and IMS-TOF MS have been combined. It has been shown that MALDI-FT-ICR MS is a helpful method when high-resolution MALDI-MS measurements are required.

Fig.6 TOF: Sample target for a MALDI mass spectrometer

 

iii. Atmospheric pressure:

The ionisation method known as matrix-assisted laser desorption/ionization (MAPLDI) at atmospheric pressure (AP) functions in a typical atmospheric environment as opposed to a vacuum. Vacuum MALDI and AP-MALDI differ primarily in the pressure at which the ions are produced. Ions are usually generated at 10 mTorr or less in vacuum MALDI, whereas atmospheric pressure is reached in AP-MALDI. The primary drawback of the AP-MALDI method in the past when compared to traditional vacuum MALDI has been its low sensitivity; nevertheless, attomole detection limits have been documented, and ions can be transferred into the mass spectrometer with great efficiency. Mass spectrometry (MS) uses AP-MALDI for a number of purposes, from proteomics to drug discovery. Proteomics, the mass analysis of DNA, RNA, PNA, lipids, oligosaccharides, phosphopeptides, bacteria, small molecules, and synthetic polymers are among the common topics covered by AP-MALDI mass spectrometry. Similar applications are also possible with vacuum MALDI instruments. An ion trap mass spectrometer or any other MS system with an electrospray ionisation (ESI) or nano ESI source can be readily connected to the AP-MALDI ion source [4]. It is known that singly-charged ions are the primary product of MALDI ionisation at low pressure (see "Ionisation mechanism" below). On the other hand, ionisation at atmospheric pressure has the ability to produce highly charged analytes, as demonstrated by infrared and subsequently nitrogen lasers. The ability to measure high-molecular-weight compounds like proteins in instruments that only offer smaller m/z detection ranges, like quadrupoles, makes multiple charging of analytes crucial. In addition to pressure, the matrix's composition plays a crucial role in producing this outcome.

iv. Aerosol:

One method of ionisation used in aerosol mass spectrometry involves aiming a laser at individual droplets. Single-particle mass spectrometers (SPMS) are the systems that go by this name. Before being aerosolized, the sample may optionally be combined with a MALDI matrix.

 

 

§  Ionization mechanism:

In the dried-droplet spot, the matrix crystals are targeted by the laser beam. The laser energy is absorbed by the matrix, and it is believed that this event primarily desorbed and ionises (by adding a proton). Numerous species can be found in the hot plume created by ablation, including matrix clusters, nanodroplets, neutral and ionised matrix molecules, and matrix molecules that have been protonated and deprotonated. Analyte ionisation may involve delayed species, however the MALDI mechanism is still up for discussion. It is then believed that the matrix charges the analyte by transferring protons to the analyte molecules (such as protein molecules). An ion that is seen after this procedure is made up of the original neutral molecule [M] plus any ions that have been added or subtracted. Quasimolecular ions are those that have an added proton (for example, [M+H] +, [M+Na] +, or [M-H] −), or an added sodium ion (for example, [M+Na] +). Depending on the type of matrix, the laser's intensity, and/or the voltage applied, MALDI can produce singly charged ions or multiply charged ions ([M+nH] n+). Keep in mind that every species listed here has an even number of electrons. For example, in the case of matrix molecules and other organic molecules, ion signals of radical cations (photoionized molecules) can be observed [4]. Primary and secondary processes leading to ionisation are postulated by the gas phase proton transfer model, which is implemented as the coupled physical and chemical dynamics (CPCD) model of UV laser MALDI. The two main processes are the initial charge separation caused by the matrix absorbing photons and the energy pooling that results in the formation of matrix ion pairs. Primary ion formation is the result of a UV photon being absorbed, which produces excited state molecules by

S₀ + hν → S₁

S₁ + S₁ → S₀ + S_n

S₁ + S_n → M⁺ + M⁻

in which Sn is a higher electronic excited state, S0 is the ground electronic state, and S1 is the first electronic excited state. Proton transfer or electron transfer ion pairs, denoted by M+ and M− above, can be the product ions. Ion-molecule interactions take place in secondary processes to create analyte ions.

Fig.7 (Ionization Mechanism) In the lucky survivor model, positive ions can be formed from highly charged clusters produced during the break-up of the matrix- and analyte-containing solid.

The cluster ionisation mechanism, also known as the lucky survivor model, proposes that analyte molecules are integrated into the matrix while retaining their charge state from the solution. When laser-ablated clusters break apart, charge separation leads to the formation of ions. The so-called lucky survivors are ions that are not neutralised by recombination with photoelectrons or counter ions. According to the thermal model, in melted matrix liquid, the high temperature promotes proton transfer between matrix and analyte. The ion-to-neutral ratio is a crucial parameter for the theoretical model's justification, and its incorrect citation could lead to an incorrect assessment of the ionisation mechanism. The ion-to-neutral ratio as a function of laser fluences and the rise in total ion intensity as a function of analyte concentration and proton affinity are both quantitatively predicted by the model. Additionally, this model implies that the primary source of metal ion adducts (such as [M+Na] + or [M+K] +) is the thermally induced dissolution of salt.

Analyte ions of volatile or non-volatile compounds can be produced using the matrix-assisted ionisation (MAI) method without the need for laser ablation, thanks to matrix preparation techniques similar to those of MALDI. Electrospray ionisation produces ions with nearly identical charge states to those produced by simply subjecting the matrix containing the analyte to the mass spectrometer's vacuum. It is suggested that this process and MALDI probably share some mechanistic similarities.

The usual estimate for ion yield is between 10−4 and 10−7, however some experiments suggest even lower yields of 10−9. Shortly after MALDI was introduced, attempts had been made to address the problem of low ion yields, including post-ionization using a second laser. The majority of these efforts had poor signal increases and only patchy success. This could be explained by the use of axial time-of-flight instruments, which expand the plume rapidly with particle velocities of up to 1000 m/s, operating at pressures in the source region of 10−5 to 10−6.

Using a modified MALDI source operated at an elevated pressure of approximately 3 mbar coupled to an orthogonal time-of-flight mass analyzer, successful laser post-ionization was reported in 2015. The laser used was wavelength-tunable, operating at a wavelength of 260 nm to 280 nm, below the two-photon ionisation threshold of the matrices used. This resulted in an increase of up to three orders of magnitude in the ion yields of several lipids and small molecules. Because of the second laser and the second MALDI-like ionisation process, this method—known as MALDI-2—was later adopted by other mass spectrometers that had low bar operating sources.

Advantages MALDI-TOF:

·      Decrease the turnaround time significantly. Processing times are comparable to those of fast biochemicals.

·      There is little sample needed, and sample preparation is easy. It only takes one colony to produce spectra that are good enough.

·      Low consumable costs and cost effectiveness.

·      Robust, automated reproducibility across labs

·      Wide range of application (all bacteria, including fungi and anaerobes)

·      An open, flexible system that the user can grow.

Disadvantages:

·      Only if the spectral database has peptide mass fingerprints of the type strains of particular genera, species, subspecies, or strains can new isolates be identified.

·      No information about susceptibility is given.

·      Unhelpful for direct clinical specimen testing (apart from urine).

·      Repeat analysis and additional processing (extraction) are necessary for certain organisms.

·      Different studies have different acceptable cutoff scores, and some closely related organisms are not distinguished.

5. IR MALDESI MS:

Since the development of electrospray ionisation (ESI) and matrix-assisted laser desorption ionisation (MALDI) sources—which are still the main techniques for ionising proteins—mass spectrometry (MS) has emerged as the principal analytical tool utilised to characterise both target and therapeutic proteins. When connected to a time-of-flight (TOF) mass spectrometer, MALDI can analyse samples quickly—at a rate of less than one second per sample—and generates mostly singly charged ions, which make it possible to determine the molecular weight directly. [16]

LC-MS systems, which are reliable and helpful for up to 384 sample analyses. However, faster and more thorough MS analysis techniques are required for some activities where 1000–10,000 samples need to be analysed.

i.                    Desorption electrospray ionization (DESI):

There have also recently been reports of native MS with DESI. Protein MS imaging in tissue has also been demonstrated using nano DESI. The first native-MS imaging of proteins using nano DESI was reported by Cooper et al.

ii.                  High Throughput Protein Analysis:

After IR-MALDESI-MS was developed for protein standard measurement, its suitability for high throughput protein analysis was examined.

iii.                BTK Phosphorylation and Compound Binding Kinetics:

After IR-MALDESI-MS was developed for protein standard measurement, its suitability for high throughput protein analysis was examined.iv. Compound Modification to La Protein and Top-down Analysis

The human La antigen (La protein) is highly reactive to thiol-reactive compounds and can be used to assess compound redox activity to identify false positives from biochemical screens.[16]

 

 

iv.                High Throughput Sample Cleanup Coupled with IR-MALDESI-MS:

Even at relatively high salt concentrations, many salts and buffers exhibit concentration-dependent ion suppression effects. Sample clean-up is necessary for certain applications due to relatively low protein concentrations and high salt/buffer concentrations. Therefore, the applicability of IR-MALDESI could be greatly increased by a quick, multiplexed sample clean up step. To obtain high-quality spectra and facilitate data interpretation, desalting had to be done. High throughput desalting of individual protein samples can be accomplished using a variety of techniques, the majority of which are marketed. Two plate-based strategies with IR-MALDESI-MS:

a)      Slit Plate:

Slit plate is a desalting plate that is sold commercially and operates on a similar concept as TopTip. Different stationary phase chromatographic materials can be inserted into micrometer-sized slits cut at the bottom of wells.

MagneticBeads:

Another quick and adaptable way to clean samples is with magnetic beads. To desalt or isolate and concentrate a protein of interest, they can be coated with a variety of materials, including protein A, Ni-NTA, and materials from reversed phase chromatography. Following protein binding, unwanted species can be eliminated by performing washes and using magnets to gather the beads. Numerous kinds of liquid handling equipment can automate the sample cleanup process.In [16]

 

6. Method:

·         Instrumentation:

A description of the high throughput IR-MALDESI-MS system was given. In short, samples were energised using a 2970 nm laser, and the neutral laser plume was ionised using an electrospray emitter that was oriented in relation to the MS inlet. The Q Exactive HF-X mass spectrometer (Thermo Fisher) was connected to the IR-MALDESI source. Standard microtiter plate analysis was made possible by an extended capillary equipped with a motorised stage and a specially designed cartridge heater.

 

MS Conditions:

With the exception of the top-down experiment, which employed a resolving power of 240, 000 (FWHM at m/z = 200), intact protein analysis was conducted using a resolving power of 7, 500 (FWHM at m/z = 200). The ESI solvent, which was 80/20 methanol/water v/v with 0.1% formic acid, received a spray voltage of 4 kV. There was a 2 µL/min ESI flow. The extended capillary's temperature was set to 120 °C, while the capillary's temperature was fixed at 400 °C.

·         BTK Experiments:

The Bruceton's tyrosine kinase (BTK) phosphorylation experiment was conducted in a buffer containing 0.5 mM MgCl2, 75 mM ammonium acetate, 5 mM HEPES pH 7.5, and 0.5 mM ATP. There was 50 µM BTK used. The BTK binding experiment was conducted in 5 mM HEPES pH 7.5. The buffer was used to dilute 10 mM compound stock solutions to the appropriate concentrations. To start the reaction, 10 µL of 35 µM BTK was first added to each well, and then an equal volume of 2X compound solution was added. The BTK compound solution received an equal volume of 1% formic acid in order to denaturize BTK. Real-time IR-MALDESI readout was done straight from the buffer.

Alarm MS:

In order to eliminate any remaining dithiothreitol from the storage buffer, the first buffer to be switched to PBS was La Protein. Before being subjected to IR-MALDESI-MS analyses, 0.16 mg/mL La protein was desalted with C4 TopTip after being incubated with 500 µM compounds for one hour.

·         Protein Desalting with BcMag Magnetic Beads:

 Magnetic beads were kept at 4 °C after being suspended in a 50 mg/mL solution of methanol and water (v/v). Plates were filled with 10 µL/well of the stock mixture, and the storage solution was removed using a magnetic carrier and a blue washer. To equilibrate the mag. beads, 30 µL/well of equilibration buffer (0.5% trifluoroacetic acid, 95/5 water/acetonitrile v/v) was added and then removed. Before being added to the working plate, 30 µL of the protein sample was combined with 10 µL of the sampling binding buffer (2% trifluoroacetic acid, 95/5 water/acetonitrile v/v) and allowed to sit for one minute. To release the protein, 30 µL of elution buffer (50/50 acetonitrile/water) was added after three rounds of washing with equilibration buffer. A PCR plate was used to elute the desalted samples for IR-MALDESI-MS analysis.

 

7. Current status of instrumentation in proteome analysis by mass spectrometry:

 Despite the remarkable rate of advancement in mass spectrometric instrumentation for protein analysis, no single instrument currently satisfies all the needs for high-throughput proteome research within a systems biology framework. There are, in fact, a staggering amount of specialised instruments available. The number of mass spectrometers that are currently on the market is increased when MALDI or ESI sources are used in conjunction with the various mass analyzer types that were previously discussed. Selecting the right technique for a given biological question requires a thorough understanding of the functional principles of mass spectrometers, as different models have different advantages and disadvantages. In an effort to clarify the best courses of action, we present here a general overview of the mass spectrometer types that are frequently employed in proteome research.

8. Applications:

Biochemistry: Strong/weak ion exchange, size exclusion chromatography, affinity chromatography, isotope-coded protein labelling (ICPL), SDS-PAGE, and two-dimensional gel electrophoresis are among the proteomics techniques that use MALDI to quickly identify proteins isolated through gel electrophoresis. The most widely used analytical application of MALDI-TOF mass spectrometers is peptide mass fingerprinting. Using high energy collision-induced dissociation or post-source decay, MALDI TOF/TOF mass spectrometers can determine the amino acid sequence of peptides (for additional uses, see mass spectrometry). MALDI-TOF has been used to characterize post-translational modifications. For example, it has been widely applied to study protein methylation and demethylation. However, care must be taken when studying post-translational modifications by MALDI-TOF. For example, it has been reported that loss of sialic acid has been identified in papers when dihydroxybenzoic acid (DHB) has been used as a matrix for MALDI MS analysis of glycosylated peptides. Using sinapinic acid, 4-HCCA, and DHB as matrices, S. Martin studied the loss of sialic acid in glycosylated peptides by metastable decay in MALDI/TOF in linear mode and reflector mode [14]. A group at Shimadzu Corporation derivatized the sialic acid by an amidation reaction as a way to improve detection sensitivity and also demonstrated that ionic liquid matrix reduces a loss of sialic acid during MALDI/TOF MS analysis of sialylated oligosaccharides. It has been determined that THAP, DHAP, and a combination of 2-aza-2-thiothymine and phenylhydrazine are matrices that may be utilised to reduce sialic acid loss during MALDI MS analysis of glycosylated peptides. It has been suggested that using IR MALDI rather than UV MALDI can reduce the loss of some post-translational modifications. In addition to proteins, lipids have also been studied using MALDI-TOF. It has been used, for instance, to research the catalytic processes of phospholipases. Apart from lipids, MALDI-TOF has also been used to characterise oligonucleotides. For instance, after oligonucleotide synthesis, a combination of spermine and 5-methoxy salicylic acid can be utilised as a matrix for oligonucleotide analysis in MALDI mass spectrometry in molecular biology.

Organic chemistry: Some synthetic macromolecules have molecular weights that extend into the thousands or tens of thousands, where most ionisation techniques have trouble producing molecular ions. Examples of these macromolecules are catenanes and rotaxanes, dendrimers and hyperbranched polymers, and other assemblies. Chemists can quickly examine and validate the outcomes of these syntheses using the straightforward and quick analytical technique MALDI.

Polymers: The molar mass distribution in polymer can be found using MALDI chemistry. Using MALDI to characterise polymers with polydispersity greater than 1.2 is challenging because of the discrimination of signal intensity against oligomers of higher mass. AgTFA or dithranol forms an excellent matrix for polymers. AgTFA must be added after the sample has been combined with dithranol; otherwise, the sample will precipitate out of the solution.

Microbiology: A common application of MALDI-TOF spectra is the identification of microorganisms like fungi and bacteria. A section of the relevant microbe's colony is deposited onto the sample target and covered in matrix. In a process known as biotyping, the mass spectra of expressed proteins are generated, examined by specialised software, and contrasted with profiles that have been stored to determine the species. It is now a standard technique for species identification in clinical microbiological laboratories and has advantages over other immunological or biochemical techniques. With an emphasis on influenza viruses specifically, the advantages of high-resolution MALDI-MS carried out on a Fourier transform ion cyclotron resonance mass spectrometry, or FT-MS, have been shown for typing and subtyping viruses through single ion detection known as prototyping [15]. Its ability to quickly and accurately identify a wide range of microorganisms at a low cost straight from the selective medium used to isolate them is one of its main advantages over other microbiological identification techniques. Turnaround times are much faster when there is no need to purify the suspect or "presumptive" colony. For instance, it has been shown that bacteria can be directly detected from blood cultures using MALDI-TOF. The ability to forecast a bacteria's susceptibility to antibiotics is an additional benefit. One mass spectral peak can forecast Staphylococcus aureus's methicillin resistance. Acinetobacter baumannii and pneumonia are among the Enterobacteriaceae resistant to carbapenems, and MALDI is also capable of detecting their carbapenemase. The majority of antibiotic resistance-mediating proteins, however, are larger than the 2000–20,000 Da range of MALDI-TOF for protein peak interpretation. In rare instances, such as the 2011 NIH Klebsiella pneumonia carbapenemase (KPC) outbreak, a correlation between a peak and a resistance-conferring protein can be established.

Parasitology: Plasmodium, Leishmania, and trypanosomatids are just a few of the parasites that can be found and identified using MALDI-TOF spectra. Apart from these unicellular parasites, parasitic insects like lice or cercariae—the free-swimming stage of trematodes—can also be identified using MALDI/TOF.

Medicine: When diagnosing illnesses, MALDI-TOF spectra are frequently used in conjunction with other analysis and spectroscopy methods. Because it can quickly identify proteins and changes to proteins without the expense or processing power of sequencing or the expertise or time required to solve a crystal structure in X-ray crystallography, MALDI/TOF is a diagnostic tool with a lot of potential.

Necrotizing enterocolitis (NEC), a terrible illness that affects the intestines of premature infants, is one instance of this. Since the symptoms of NEC and sepsis are so similar, many infants pass away while they are being diagnosed and treated. To identify the bacteria found in the faeces of infants who tested positive for NEC, MALDI/TOF was utilised. This study did not address the disease's mechanism, instead concentrating on characterising the faecal microbiota linked to non-epidermal cystic acne. It is hoped that a similar method could be applied as a fast, non-sequencing diagnostic tool. The field of cancer is one more instance of MALDI/TOF's diagnostic efficacy. One of the deadliest and most challenging cancers to diagnose is still pancreatic cancer. Pancreatic cancer has long been thought to be caused in part by impaired cellular signalling brought on by mutations in membrane proteins. A membrane protein linked to pancreatic cancer has been found using MALDI/TOF, which at one point may even be used as an early detection method [15]. In addition to dictating diagnosis, MALDI/TOF may also be used for treatment planning. One technique for assessing bacterial drug resistance, particularly to β-lactams (a class of antibiotics), is MALDI/TOF. Drug resistance to common antibiotics is indicated by the presence of carbapenemases, which is detected by the MALDI/TOF. This has the potential to be a three-hour turnaround time for determining whether a bacterium is drug-resistant. This method may assist doctors in determining whether to start off with more potent antibiotic prescriptions.

Detection of protein complexes: Investigations of large protein complexes using MALDI-MS have been reported, following preliminary findings that some peptide-peptide complexes could survive MALDI deposition and ionisation.

 

CONCLUSION:

In a systems biology setting, mass spectrometry-based protein identification is currently the most potent tool available for proteome research. The range and continuous advancement of mass spectrometric methods ensures continued enhancements in the efficiency of protein identification and expands the application of proteomics analyses in general. Since mass spectrometric protein analysis is currently in such a dynamic state, it is challenging to create the long-needed standards for widely recognised practises. As such, a direct comparison between disparate instruments lacks significance. Developing effective methods for protein detection or quantification requires a basic understanding of the various mass spectrometric designs. Still, it is currently unclear which design will gain widespread acceptance. With its latest improvements, FT-ICR-MS is now the most promising MS platform available; however, it is important to monitor other developments as well. Among them are the new ion trap designs, such as the linear ion trap and Orbitrap, which now form the basis of a new creation of extremely sensitive tandem mass spectrometers with high power. These mass analyzers could offer an affordable and space-saving substitute for FT-ICR-MS systems. Thus, there is a greater chance that various MS technologies will continue to coexist.

 In proteome analysis, the single mass analyzer design which cannot perform "real" tandem MS—is in danger of becoming extinct. The requirement for higher sample throughput in conjunction with high-performance MS may be the most difficult of all. When combined with isotope tagging techniques, automated multi-dimensional peptide separation techniques should offer mass spectrometry a high throughput platform with enough analytical depth for proteome analyses.

Increased automation of mass spectrometric analysis, proteome interpretation, and sample handling is producing a deluge of quantitative and qualitative proteome data, as has already occurred in genomics. It is increasingly clear that the primary impediment to mass spectrometric protein identification is the high-performance computation of recorded MS data. [16]

 

ACKNOWLEDGMENT

Authors are thankful to Principal and Management J.I.I.U' S Ali Allana College of Pharmacy Akkalkuwa, Dist. Nandurbar for providing moral support and necessary facilities for completion of this work.

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