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[Review article]

Major Applications of Mass- Spectrometry

Submitted by

Subhadip chakraborty

22MBT10128

Course code:- 22BTT-604

Under the supervision of

Dr. Vagish Dwibedi

UIBT

Chandigarh university
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Abstract:- By measuring the mass-to-charge ratio of ions in the gas phase, mass
spectrometry is a technique for figuring out the molecular mass. By causing either a
charge loss or gain, the ions are produced in the ionisation source throughout this
process (e.g. electron ejection,protonation or deprotonation). The ions can be
electrostatically steered into a mass analyzer once they have generated in the gas phase,
separated based on mass, and then detected. Mass-spectrometry can be used for various
purposes like, in biological research, for metabolite identification, for quantitation of
DNA, in the study of genomics and proteomics, in biomarker discovery and also in
newborn screening etc. Mass spectrometry can be used to identify unknown
compounds, quantify known compounds, and determine the structural and chemical
properties of substances. Mass spectrometry works by ionization and determination of
masses of charged analyte molecules. Mass spectrometry is a technique that is actually
boon to science.Mass spectrometry combined with various other methods can be used
for various purposes like for e.g. FT-ICR-MS with other mass spectrometric methods
are used for derivatization of oligosaccharides and glycoconjugates that helps to detect
sensitivity and informative fragmentation. The introduction of Tandem-Mass
spectrometry has led to rapid expansion into new fields of science and research. After
years of scientific progress, Mass spectrometry can also be used for diagnosis and
screening of various disorders in neonates and adults.Every year, Indian Society for
Mass Spectrometry(ISMAS) conducts a workshop on Mass spectroscopy technique and
nowadays in research ,the application of this technique is increasing.

Keywords:- Mass spectrometry, Application of Mass spectrometry in

biological research, Mass spectrometry in food analysis, MS uses in
metabolite identification,Mass spectrometry and proteomics
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1. Introduction:-

J.J. Thomson first created the mass spectrometer in 1900, that used fixed magnetic and electric fields to separate
ions of various masses and energies. He realised that charged particles with varied momentum exhibited different
behaviours in an electric field and utilised this fact to separate ions with various masses[Dubey, 2018].Its
substantial usage for study in numerous biological science fields was initiated in the 1980s. Mass spectrometry
emerged as a crucial tool for genomes and proteomics research between 1990 and 2000.When used with mass
spectrometry, 2D electrophoresis has greater power[Banwell and McCASH, 2013]. The unidentified protein spot
is removed from the gel, trypsinized, and broken up into pieces. These fragments are then examined by mass
spectrometry, and their masses are plotted[Hoffman and Stroobant, 2007]. The estimation of this mass
fingerprint is possible. By measuring the mass-to-charge ratio of ions in the gas phase, mass spectrometry is a
technique for figuring out the molecular mass. By causing either a charge loss or gain, the ions are produced in
the ionisation source throughout this process (e.g. electron ejection,protonation or deprotonation)[Domon and
Aebersold, 2006]. The ions can be electrostatically driven into a mass analyzer, separated based on mass, and
then detected after they have formed in the gas phase[Biemann, 1961]. By measuring the mass-to-charge ratio of
ions in the gas phase, mass spectrometry is a technique for figuring out the molecular mass. By causing either a
charge loss or gain, the ions are produced in the ionisation source throughout this process (e.g. electron
ejection,protonation or deprotonation)[ Ham et al., 2000]. The ions can be electrostatically driven into a mass
analyzer, separated based on mass, and then detected after they have formed in the gas phase. There are three
main parts of a mass spectrometer: the ionisation source, the mass analyser, and the detector. Ionization sources
involve the gaseous ionisation of the analyte, or target molecule, for analysis[Anson, 1999]. Molecular ions are
created when the molecules receive or lose a charge (via electron ejection, protonation, or deprotonation). The
ions are produced and separated in the analyzer based on their mass-to-charge (m/z) ratio[Renner et al., 2000].
After mass spectrometry of the analyte, the mass spectra acquired in the detector are used to calculate the
molecular masses of the separated ions[Singer et al., . Proteases are responsible for breaking down proteins into
peptides (e.g. trypsin). Liquid chromatography is used to separate the peptides (such anion exchange affinity or
reverse phase column chromatography). If proteins are broken down into peptides, they can be recognised
quickly[Doerge et al., 2000].

2. Applications of Mass-spectrometry:-

In order for mass spectrometry to function, polar charged biomolecules like peptides, proteins, or nucleic acids
must first be converted into gas phase ions. When the sample (M) is introduced into the mass spectrometer, it
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goes through ionization[Andrey, 2003]. Proteins can undergo protonation at a variety of locations. The side chain
amino group and all of the backbone amide nitrogen may be protonated[Biemann, 1962].

Electrostatic force propels the charged molecules into the analyzer. The ions are separated by the analyser based
on the m/z ratio. The detector locates the ions and sends the signals to a computer[Burlingame et al., 1996]. The
computer stores and processes the information[Mano and Goto, 2003]. The following processes are involved in
the functioning of the mass spectrometer: (i) Producing the sample in an ionised form in the gas phase, (ii). Ions
are accelerated in an electric field, with the velocity of each ion emerging being proportional to its mass-to-
charge ratio (m/z), (iii). The ions' entry into an area without a field and (iv). Determining the ions' arrival times,
with the ions' time-of-flight giving their mass-to-charge ratio[Grayson, 2003].

2.1. Applications of Mass spectrometry in biological research:-

There are many different MS uses in biology. They can be grouped into three fundamental types: isotope ratio,
macromolecular, and small organic molecule mass spectrometry. The relative stable isotopic abundance of
elements can be determined using the isotope-ratio MS (IRMS) method[Finehout and Lee, 2003]. IRMS is often
utilised in projects utilising stable isotopic tracers in biomedical research. Compounds containing elements with
unusual stable isotope ratios are added to a system of interest by feeding or by injection[Willard et al., 1988] .
IRMS is then used to track the change in the element's isotopic ratio in the system. Additionally, stable isotope
tracers can be utilised as a clinical tool for disease detection or to examine energy expenditure[Guo, 1999].
Faraday cups are used as the ion detectors and magnetic sectors are used as the mass analyzers in IRMS. In
addition, MS is an useful method for the high-throughput study of chemicals derived from combinatorial
libraries, such as those used to find novel medications[Laeter, 2001]. Both structural data and details about a
molecule's binding affinity can be obtained if the mass spectrometer is coupled to an LC system with the right
columns. The major metabolites of drugs can be identified from in vitro investigations using MS, and their in
vivo measurements can be utilised to estimate pharmacokinetic parameters. MS can also be used to examine
DNA methylation and other changes[Gyagi et al., 2002].The structure of a wide variety of oligosaccharides can
be estimated by tandem Mass-spectrometry. MS is used to assess the quantity and duration of fatty acylcarnitines
in the blood. Doctors can identify the presence of a fatty acid oxidation disorder and, in certain cases, the
absence of that particular enzyme can be identified which is absent in the child, by comparing the resulting
spectrum to that of a healthy infant[Patterson and Veillon, 2001].Today a crucial tool for proteome analysis is
MS. It can reveal informations about a protein's identification, its quantity, and any alterations the protein have.
One can determine the original amino acid sequence of the peptide[Mano and Goto, 2003]. The precise location
of the alteration can then be identified by doing an MS/MS analysis on the phosphorylated peptide. With the help
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of MS/MS analysis, the parent protein and the location of phosphorylation may be determined from the changed
peptides[Barber et al., 1981]. The mass spectrometer's many scanning modes can also be used to detect the
presence of phosphorylation. A wide variety of post-translational changes have been studied using MS[Fenn et
al., 1989]. It should be emphasised that the structure of changes made by heterogeneous molecules (such
glycans) could also be examined using MS[Tanaka et al., 1988].

2.2 Applications of Mass-spectrometry for metabolite identification:-

The majority of analytical methods used for metabolite identification involve high performance liquid
chromatography (HPLC) in combination with mass spectrometry (MS). Due principally to the discovery and
commercialization of air pressure ionisation (API) sources, which made it possible to directly connect liquid
chromatography (LC) with MS, mass spectrometry (MS) has become the premier analytical instrument for the
detection and identification of metabolites like it can be used for identification and structural determination of
flavonoid glycosides[Ma et al., 2015]. Identification and structural analysis of flavonoid glycosides can be done
using mass spectrometric methods. The application of several physical principles, both for sample ionisation and
separation of the ions produced in accordance with their mass (m) to charge (z) ratio (m/z), is one of the
distinguishing characteristics of MS[Stobiecki, 1999]. MS offers far more versatility in the detection,
quantification, identification, and structural characterization of compounds than other spectroscopic techniques
and the advent of so-called "soft" ionisation techniques has led to a rise in the use of MS for the investigation of
flavonoid glycosides[Baldi et al., 1995]. By combining tandem MS with collision-induced decomposition (CID
MS/MS) and desorption ionisation techniques, the structural information of different metabolites can be
improved. O- and C-glycosylated flavonoids have been studied structurally using EI mass spectrometry to some
extent. Field desorbtion technique can be used for analysis of intact flavonoid glycosides(Geiger and Schwinger,
1980). Field desorption mass spectrometry was also employed during research on acylated glycosides from
Bryum cappilare to confirm the replacement of the sugar moiety with a malonyl group[Stein et al., 1985].The
combination of liquid chromatography and mass spectrometry (LC-MS), has excellent sensitivity and selectivity
that can separate, detect, and identify a wide variety of metabolites in the presence of endogenous
material[Caldwell et al., 1995]. Different mass spectrometric techniques (LSIMS normal and CID linked scan
spectra, EI GC/MS and others) were used for structural analyses of flavonoid glycosides isolated from Lupinus
luteus(Franski et al., 1999). Each drug-derived entity's molecular weight can be easily calculated from the mass-
to-charge ratio (m/z) of the protonated or deprotonated molecules recorded by LC-MS[Wang et al., 2005]. Fast
atom bombardment (FAB) and liquid secondary mass spectrometry (LSIMS) can also be used for analysis of
flavonoid glycosides. Detail structural information of metabolites can be obtained from LCMS/ MS
investigations by carefully examining the fragmentation products of protonated or deprotonated molecules.
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Before any analytes can be detected by a mass spectrometer, they must be transformed into gas-phase ions in an
ionization source[Raffaelli and Saba, 2003]. For the analysis of plant secondary metabolites, liquid
chromatography using CF-FAB or CF-LSIMS interfaces has also been used for analysing profiles of acylated
isoflavonoid glycosides present in alfalfa (Medicago sativa ) and chick pea (Cicer arientium ). LC-ESI/MS and
LC-ESI/MS/MS can be used for recognizing anthocyanin glycosides in plant tissue and cell cultures of Daucus
carota[Mauri et al., 1999]. The LC-ESI/MS method was also used for a selective screening of 6'-O-malonylated
or acetylated glucoconjugates found in plant tissues (fruits, roots, and leaves) of species that are consumed by
humans, as well as in foods made from soy and tomatoes[Barnes et al., 1994]. Large peptides and proteins'
molecular weights can be precisely estimated from the observed m/z of their multiply-charged ions when a mass
spectrometer is connected to an ESI source[Dams et al., 2003]. The main method for finding metabolites in
intricate biological matrices is tandem mass spectrometry (MS/MS). Today, LC-MS is often used for
characterization and identification of metabolites from biological matrices. LC-TOFMS systems' excellent
sensitivity and high mass accuracy will ensure that they continue to play a significant role in metabolite
identification[Geyer et al., 2004]. LITs and hybrid LITs will become more useful for rapid metabolite
characterisation through data-dependent acquisition as a result of their high sensitivity and quick scan speed,
enabling numerous scans in MS and MS/MS modes to be carried out automatically. Consideration should be
given to using LC-NMR/MS as a reliable method for accurately identifying metabolites that have been detected
from biological matrices or their extracts[Kauppila et al., 2002]. Recently, metabolites from compounds
containing bromine were quantified using liquid chromatography(LC) and on-line inductively coupled plasma
mass spectrometry (LC-ICPMS)[Rauha et al., 2001]. If LC-CRIMS (chemical reaction interface to mass
spectrometry) technology were to become widely available, it could be possible to start drug metabolic
investigations much earlier in the clinical development process than with radioisotopes[Yang and Henion, 2002].

2.3. Applications of Mass- spectrometry for quantitation of DNA adducts and

for solving structural problems of nucleosides:-

A DNA adduct is a piece of DNA joined to a substance that causes cancer. Carcinogenesis, the growth of
malignant cells, may result from this process of formation of DNA adduct. DNA adducts are helpful for
calculating how much of a carcinogen an organism has been exposed to[Koc and Swenberg, 2002]. When free
radicals or electrophilic molecules attack DNA, DNA adducts are generated. The most widely used technique for
quantifying DNA adducts is 32P-postlabeling. DNA adducts are good indicators for assessing the degree of
genetic material damage, which has long been important for understanding the mechanism of carcinogenesis and
calculating the risk of cancer[Holt et al., 1998]. DNA adducts are crucial agents for the development of particular
biomarkers and can be used for understanding the mechanism of carcinogenesis[Muller et al., 1997]. Electron
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capture- mass spectrometry(EC-MS) can be used for the analysis of DNA adducts. There are three main
processes in the quantification of DNA adducts by mass spectrometry: DNA isolation, DNA hydrolysis, and
enrichment of adducts of interest in the hydrolysate, followed by mass spectrometric analysis by LC-MS or GC-
MS[Rouzer et al., 1997]. Mass spectrometry is a quantitative method that provides high level of specificity in the
detection process of DNA adducts at a selected mode known as ‘selected ion monitoring’. In high resolution
mass spectrometry and tandem mass spectrometry method, a mode named ‘selected reaction monitoring’(SRM)
can be used for specific detection of DNA adducts[Doerge et al., 1998].

DNA isolation from tissue

Spike with internal standard

Hydrolysis/Digestion of DNA

Enrichment of DNA adduct of interest

Quantitation by Mass- spectrometry

Fig.:- Schematic representation of DNA adduct quantification by Mass-

spectrometry[Source:- Koc and Swenberg, 2002]

The most commonly used mass-spectrometry methods that are used for quantitation of DNA adducts nowadays
are GC-MS, LC-MS, capillary zone electrophoresis-Mass spectrometer(CE-MS), gas chromatography with
negative ion chemical ionization mass spectrometry(GC-EC-MS) and liquid chromatography with Electrospray
mass spectrometry(LC-ESI-MS)[Hakala et al., 1999]. Reactive oxygen species are free radicals formed during
metabolic pathways that can also generate DNA adduct such as 8-OH-Gua and can be quantified using GC-MS
methods. Except GC-MS, LC-MS methods are also well suited for 8-OH-Gua quantitation[Casale et al., 2001].
Ethenobases are exocyclic adducts that are generated when DNA bases combine with endogenous metabolites
of lipid peroxidation products or with vinyl chloride or urethane. 1,N6-ethenoadenine (1,N -ϵ Ade), 3,N4 -
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ethenocytidine (3,N4 -ϵCyt), 1,N2 -ethenoguanine (1,N2 -ϵ Gua), and N2,3-ethenoguanine(N2 ,3-ϵGua) are four
ethenobases that are widely known[Koc and Swenberg, 1999]. For the purpose of quantifying N 2,3-εGua in
preweanling and adult Sprague- Dawly rats, Fedtke et al. created the gas chromatography negative ion chemical
ionisation isotope dilution mass spectrometry (GC-EC-IDMS) method. Both GC-EC-IDMS and LC-ESI-IDMS-
MS techniques for quantifying 1,N -ϵAde in human placental DNA have been invented by Chen et al. . One of
the main endogenously generated aldehydic chemicals upon peroxidation of membrane lipids is,
malondialdehyde (MDA). By utilising electron capture mass spectrometry, Chaudhary et al. were the first to
establish a quantitative assay for measurement of a cyclic adduct generated by interaction of MDA with N1 and
N2 of guanine, pyrimido[1,2-α]purin-10(3H)-one (M1G). Due to their prevalence in cigarette smoke and
contaminated air, polycyclic aromatic hydrocarbons (PAH) are a significant class of substances that endanger
human health. For measuring DNA adducts related to the PAHs, techniques combining capillary
electrochromatography and mass spectrometry have been used[Lawly, 1984]. Electrophore labelling has been
used by Giese et al. to quantify DNA adducts of PAH using GC-MS. For quantifying BPDE adducts with
deoxyguanosine monophosphate, Barry et al. used CE-ESI-MS-MS. LC-ESI-IDMS-MS method can be applied
for quantitation of 7HEG in human and rodent DNA[Friesen, 1991]. Mass- spectrometry can be used to define
and determine the structure of nucleosides and related compounds, especially for synthesizing the
analogs[BIEMANN and McCLOSKEY, 1962].
Table:- Applications of Mass- spectrometry in quantitation of DNA adducts

Compound Adduct Methods for detection References

1. Reactive oxygen 8-OH-Gua, 8-OH-dGuo, GC-IDMS,LC-ESI-MS- [Singer and
8-OH-dGuo, 8-OH-dAdo
species MS,LC-ESI-IDMS-MS Grunberger, 1983]
2. Lipid peroxidation N2-3ϵ-Gua,1, N2-ϵ-Gua,OH- GC-EC-IDMS,LC-ESI-IDMS- [Phillips et al., 2000]
products Ethano-Gua, 1, N6-ϵAde MS,LC-ESI-IDMS
3. Alkylating agent N3-alkyladenines GC-IDMS-MS [Ravanat et al.,
1998]
4. Radiation ThdGly, 5-OH-dUrd LC-ESI-IDMS-MS [Roberts et al., 2001]
5. Mephalan dAmp adducts of mephalan LC-ESI-MS-MS [Ham et al., 2000]

2.4 Applications of Mass-spectrometry for analysis of food related compounds:-

The importance of mass spectrometry in food research is well established. Mass spectrometry can be used for
quality control analysis of food. Liquid chromatography coupled to mass spectrometry can be used as an useful
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tool for analysis of food.Mass spectrometry is also a significant tool for protein and peptide
characterization[Careri et al., 2002]. ICP-MS, also known as inductively coupled plasma-mass spectrometry, is
an analytical method having numerous uses in the measurement of inorganic chemicals in food. Research and
development in food processing places a priority on lipid analysis[Nelson et al., 1997]. HPLC-MS and GC-MS
can be used to analyse different oil systems and fats. HPLC-APCI-MS can be used for characterizing different
fat triacylglycerols present in bovine milk[Fontecha et al., 2000].GC-MS can be used for analyzing the presence
of sterols in vegetable oils and plant samples. Polyglycerol fatty acid esters(PGE) are used in food industry as
food preservative, fat replacers and for stabilizing food emulsions[Careri et al., 1999]. Degree of polymerization
of PGE can be measured with the help of HPLC-MS method. Presence of carotene and xanthophylls can be
determined by using HPLC-MS method on the basis of particle beam interface under electron capture negative
ion (NI) method[Catinella et al., 1996]. All fat and oils contain a number of phospholipids that can be analyzed
by ESI-MS. Soyabean, egg yolk and bovine liver contain phosphatidylcholine molecules that can be converted in
Di-acylglycerol and can be identified by LC-MS techniques[Camafeita et al., 1998] . MALDI-TOF-MS was
effective in determining if cow milk was present in raw ewe and buffalo milk samples as well as in identifying
the addition of powdered milk to fresh raw milk samples[Sabbadin et al., 1999].ESI-MS with quadrupole
instrument can be used for mass determination of food and milk proteins. Carbohydrate molecule can be
characterized by using MALDI-TOF-MS and HPLC-MS through isotopic measurement of non-volatile species
of carbohydrates[Sforza et al., 2001].HPLC-ESI-MS can be used for identification of limonoid glucosides that
can be used as food additives.EI- mass spectra can be used for characterization of fat soluble vitamins such as
Vitamin D3[Trujillo et al., 2000].Oligosaccharides and their conjugates have different isomers that can be
identified using MALDI-TOF-MS analysis[Costello, 1999].FT-ICR-MS with other mass spectrometric methods
are used for derivatization of oligosaccharides and glycoconjugates that helps to detect sensitivity and
informative fragmentation[Gaskell, 1997].IR MALDI can be used for large oligonucleotide weight
determination, synthetic spectra determination and analysis of plasmid DNA and RNA transcripts upto 2180
nucleotides[Nelson, 1997].HPLC-MS can be used for characterization of various classes of biomolecules.
Proteomics and peptidomics, or the study areas that involve the evaluation of the complete pattern of proteins or
peptides in a sample, play a significant role in LC/MS. More and more peptidomics and proteomics methods
based on MS are being used in the examination of food[Stefano et al., 2012]. One of the most effective tools for
analysing the lipid components in food is HPLC-MS[Malik et al., 2010]. Because of the complexity of lipids,
two-dimensional HPLC is frequently coupled online to MS to acquire enough selectivity to enable structure
study of individual components[German et al., 2007]. Analysis of numerous lipids, primarily using HPLC-MS,
has been described by scientists, including triacylglycerols, fatty acids, carotenoids, and phospholipids collected
from a variety of meals[Steinmann and Ganzera, 2011]. Many food stuff with high functional value can be
analyzed by HPLC-MS analysis. Traditionally, GC-MS has been used to study monosaccharides and tiny
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oligosaccharides. Although it has advantages for small oligosaccharides, HPLC-MS allows for the analysis of
bigger oligosaccharides and molecular conjugates[March et al., 2006]. Without the need for time-consuming and
arduous separation, purification, and derivatization, the HPLC-MS approach enables quick identification of
unknown carbohydrates.Carbohydrates are often ionised using ESI rather than APCI due to their strong polarity
and low volatility[Sancho et al., 2005]. The best analytical method for analysing vitamins is HPLC, especially
when paired with MS. APCI ionisation is occasionally utilised for HPLC-MS, which typically uses
electrospray[Sforza et al., 2006]. There are numerous instances when vitamins in food have been analysed and
quantified using HPLC-MS. UHPLC-MS analysis, which offers superior chromatographic resolution and higher
throughput, has typically replaced HPLC-MS in recent years[Cunha et al., 2007]. Due to the numerous ways in
which they can take structural forms, (U)HPLC-MS is also frequently used to evaluate carotenoids. Since apolar
molecules make up the majority of carotenoids, APCI (and potentially APPI) ionisation may be preferable to the
more often utilised ESI. By using specialised MS/MS analysis, the aglycone portion of flavonoid O-glycosides
can be identified (so-called MSn spectra)[Mora et al., 2009]. Higher fragmentation energy may be applied, which
could result in MS/MS fragments that are unique to the aglycone. Mycotoxins can be identified by HPLC-MS
analysis[Masottiet al., 2010]. Another method to improve selectivity is pesticide analysis using high mass
resolution (HRMS), in addition to tandem mass spectrometry[Cunsolo et al., 2005]. Time of flight (TOF),
Orbitrap, or Fourier transform (FT) mass spectrometers can all be used to carry out this. A new problem in food
science and technology is food allergy caused by food allergens. Major peanut allergens in food matrices were
successfully identified and quantified using HPLC-MS/MS at the low ppm level[Fenoll et al., 2011]. HPLC-MS
is a popular method for monitoring food additives. Characterization of non-volatile components, such as
peptides, is frequently accomplished using liquid chromatography coupled with ESI mass spectrometry. In hams,
extensive peptide characterisation is typically carried out using HPLC-ESI-MS methods[Chen et al., 2004].
Protein biomarkers and quality can be identified by mass spectrometry-based proteomic analysis of hams, which
demonstrates that distinct protein patterns are associated to technical treatments. It is effective to evaluate cheese
safety using mass spectrometry[Losito et al., 2007]. The use of HPLC-MS techniques enables the precise
quantification of food poisons at very low concentrations. Mass spectrometry, in combination with
electrophoretic or chromatographic separation techniques, is a crucial instrument that has been widely used to
examine milk and its derivative products due to the complexity of the milk matrix[Weber et al., 2006]. Due to
their considerable complexity as natural products, grapes and wine are frequently and widely used as subjects for
chemical analysis. Mass spectrometry, particularly GC-MS and HPLC-MS, is frequently employed for wine
analysis[Conte et al., 2002]. HPLC-MS is mostly utilised to investigate non-volatile components, while GC-MS
is particularly significant for studying tastes. Grape polyphenols (anthocyanins, flavonols, tannins, etc.) enable
colour characterization and give insight into the mechanisms that wines use to stabilise their colour[Mills et al.,
2004]. Numerous peptides in Champagne and Sauvignon Blanc wines were detected by HPLC-MS and nano-
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HPLC-MS techniques. Ochratoxin A, a mycotoxin that is highly toxic and is frequently found in wines, is easily
detected by LC/MS analysis of wines[Shewry et al., 2003].

2.5. Applications of Mass- spectrometry in study of Proteomics, lipidomics and

metabolomics:-

The entire complement of proteins present in a cell or organism at any one time is known as the proteome, and
proteomics is the study of the proteome. An organism's proteome, or even the proteome of just one type of cell,
is significantly more intricate than the matching genome[Domon and Aebersold, 2006] . This is mainly because
practically all proteins are subject to post-translational modifications and the changes that can be introduced
through alternative splicing. The content of the proteome varies from cell to cell and occasionally depending on
the healthy or pathological status of cells or organisms[Lescuyer et al., 2007]. Our understanding of
physiological and pathological states, biological diseases or disorders, and complete proteomes can be built on
the characterization of proteins[Weinberger et al., 2002]. The hundreds of proteins involved in fundamental
physiological processes have been better identified and quantified because to ongoing improvements in mass
spectrometry (MS) instruments and methodologies[Hoffman and Stroobant, 2007]. As a result, MS-based
proteomics has been widely used to analyse biological samples and has significantly improved our knowledge of
protein functions, interactions, and dynamics, enhancing our understanding of cellular processes as well as the
physiology and pathology of the human body.In most proteomics study, MALDI and ESI are mainly used[[Issaq
et al., 2003]. The determination of a protein's molecular weight (MW) and main amino acid sequence is the
initial step in the identification and characterisation of the protein. There are two main strategies, the analysis of
intact proteins (top-down approach) and the analysis of a peptide mixture from a digested protein are both
methods for characterising proteins using mass spectrometry (bottom-up approach)[Abate et al., 2010]. The top-
down method includes ionising intact proteins in the gas-phase, followed by high-resolution mass spectrometry
without any preliminary digestion. For precise measurements, biological samples must be divided and/or
fractionated into simpler protein mixtures or a single protein[Rodriguez et al., 2007]. Shotgun proteomics is a
bottom-up form of this technique in which the protein mixture is immediately digested into a collection of
peptides, which are then separated using one or more dimensions of chromatography and examined using
tandem MS (MS/MS)[Yates et al., 2009]. It is difficult to identify and characterise glycoproteins because the
glycosylation machinery and functioning mix together a variety of glycosylated variants with variable glycan
compositions.Precise mass measurements are essential for the accurate computation of glycan composition in
MS-based proteomics[Wu et al., 2009].Mass spectrometry can also be used for identification of peptide
phosphorylation. Protein-protein interactions are essential for biological function of cell. When defect occurs in
protein-protein interaction, disease progression occurs[Leymarie and Zaia, 2012]. Several mass spectrometry
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methods (such as Affinity purification-mass spectrometry, AP-MS, ESI-MS) can be used to identify this
defects[Sokolowska et al., 2013]. Ultra-performance liquid chromatography (UPLC) and hydrophobic interaction
liquid chromatography (HILIC) combined with MS can be used metabolomic studies. UPLC-MS can be used for
metabolic analysis from liver samples[Masson et al., 2010]. The preferred analytical technique for plant
metabolomics is GC-MS[Griffin & Shockcor, 2004]. In human plasma samples, Lee et al. employed CE-ESI-MS
to examine glutathione metabolites, while CE-TOF-MS was used to investigate the metabolic profile of urine
samples from mice with accelerated ageing (Nevedomskaya et al., 2010). When NMR is coupled to LC-MS, that
technique can be used for metabolomics research. An essential technique for the rapidly growing field of
lipidomics is the mass spectrometric measurement of cellular lipids[Yang et al., 2009]. MS techniques are crucial
for lipid characterization, identification, and quantitation in the development of lipidomics[Kishimoto et al.,
2001]. When ESI-MS is used for lipid analysis, the spectrum shows the lipids' molecular ions and it can be used
for analyzing cellular lipidome. By using negative-ion ESI-MS, anionic lipids are directly and deliberately
ionised from the diluted lipid solution[Han and Jiang, 2009]. Under various spraying solution conditions, the
inclusive sets of mass spectra can be used by ESI-MS to identify each category of lipids in any biological
material. The most widely used technique for quantitative analysis of specific lipid molecular species at the
moment is ESI-MS[Wheelock et al., 2009]. Three ESIMS-based methods for lipidomics are LC-MS, tandem MS
(MS/MS), and multidimensional MS. By including a lipid extract from a biological sample without first
performing chromatographic separation, MS/MS and MDMS) are established and may be utilised as global
assessments of individual lipid species[Ivanova et al., 2009]. For the investigation of lipid species, MALDI-MS
has numerous benefits, including quickness, ease of use, high sensitivity (pmol), repeatability (samples can
frequently be tested again at a later period), and high throughput[Wenk, 2005]. The imaging of peptides,
proteins, drugs, and drug metabolites using MALDI imaging mass spectrometry (MALDI-IMS) has been
effectively used to ascertain the distribution and relative concentration of these substances[Divito et al., 2012].
MALDI-IMS technique can also be used lipid profiling of single cells. In addition to the other approaches
already in use, IM-MS can separate and characterise the complex lipid mixtures found in biological
materials[Perera et al., 2012]. Analysis of intricate biological extracts without any prior fractination served as a
demonstration of the use of IM-MS for the identification and measurement of glycerophospholipid
isomers[Wang et al., 2015].

3. Conclusion:-

The molecular weight of biomolecules, the sequencing of nucleic acids and polypeptides, and the determination
of protein structure are all determined using mass spectrometry. The benefit of this approach is that analysis may
be completed rapidly and fully automatically. The molecular structure of the parent ion can be determined from
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the mass spectrum of the fragment. For instance, peptides typically break apart along the peptide backbone at the
amide bond, forming a ladder-like arrangement of ions that reveals the order of the amino acids. Database
searches are done using the sequence. The analysis of several peptides from a single protein increases the
certainty of protein identification. The investigation of anticancer metallodrugs in intricate biological samples
and the molecular characterization of their interactions with prospective targets have made mass spectrometry
(MS) an essential instrument. For the precise identification of histone post-translational changes, mass
spectrometry is a particularly effective method. Additionally, tandem mass spectrometry of the intact proteins,
peptide mass fingerprinting after proteolysis, or a combination of these two methods can be used to pinpoint the
locations of these alterations. The benefit of this method is that analysis may be completed rapidly and fully
automatically. The molecular structure of the parent ion can be determined from the mass spectrum of the
fragment. For instance, peptides typically break apart along the peptide backbone at the amide bond, forming a
ladder-like arrangement of ions that reveals the order of the amino acids. Database searches are done using the
sequence. The analysis of several peptides from a single protein increases the certainty of protein identification.

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