What is mass spectrometry and how does it work?

Running head: DIAGNOSTIC CHEMISTRY
MASS SPECTROMETRY
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DIAGNOSTIC CHEMISTRY
Chapter 2
Mass spectrometry (MS) is required to detect the mass-to-charge ratio and MALDI-TOF
MS supplies a proper idea regarding accurate, rapid and sensitive spectra of the bio-analytes in a
sample. MALDI is defined as ionisation technique where there is absorbtion of energy through
matrix from ultraviolet lasers in creating ions from big molecules with less fragmentation. ESI
MS is another technique that deals with ligand interaction study in multiprotein, protein, RNA
and DNA system proides system provides information regarding binding specificity, affinity,
stoichiometry, stability in gas-phase of ligand target complexes as well as dissociation constant
(Harrison 2018).The main difference between MALDI and ESI is the state where the samples are
aquainted with ion source. A solvated sample is used by the sample that is permeated inside the
instruement but MALDI uses solid state.
MS is used in combination with a variety of separation methods. The two principal
methodologies for charecterisation and identification of protein by using MS are bottom-up and
top-down. Bottom-up proteomics analysis peptides through proteolytic digestion whereas top-
down analysis leads to protein intact. Bottom-up proteomics includes proteolytic digestion of
proteins proior to study by mass spectrometry. Bottom-up recommends that data regarding the
component proteins is recreated from independently recognised fragments of peptides.
Bottom-up proteomics is highly established and most extensively used method for protein
classification and identification having the capability to accomplish high-resolution separations.
Top down proteomics classifies proteins from complex biological systems. This process
characteristically permits approximately 100% sequence coverage as well as full description of
proteoforms. The precise molecular style of the protein consequential from blend of alternative
splicing, post-translational modifications and genetic variation. Disintegrated pieces for tandem

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mass spectrometry is obtained by electron-transfer dissociation (ETD) and electron-capture
dissociation (ECD). Proteins are ionised classically by electrospray ionisation as well as
entombed in quadrupole ion trap mass spectrometer or in fourier transform ion cyclotron
resonance for analysis.
The de novo peptide sequencing is a technique for performing peptide sequencing
without having any previous information of the amino acid sequence. This method is used to
obtain the peptide sequences without having any protein database that can overcome the
restrictions of peptide mass fingerprinting (PMF) that are database-dependent (Wilhelm et al.
2014). On other hand, PMF helps in protein identification where the mass of an
unidentified protein is determined. It is executed with Matrix-assisted laser or
by desorption ionization time of flight (MALDI-TOF) mass spectrometry.
2.1 Protonated peptides in mass spectrometry
‘Mobile Proton’ model suggested that peptide fragmentations are used in considering the
MS/MS spectra of protonated peptide depending on the qualitative manner by using rational
difference that is observed in case of a low-energy collision that induces dissociation for peptide
ions for lacking or having mobile proton.
2.1.1. Amino acids and peptides
Peptides and amino acids are vital components of the cell, which carries out necessary
biological functions. Amino acids are the structural unit of the cell, giving structure and shape by
responding to the signals that are transmitted from the extracellular environment (Marshall and
Verdun 2016). Certain kinds of the peptide have an essential role in coordinating activities of

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various molecules. Structure of peptides and amino acids are similar as they are joined together
by peptide bonds.
2.1.2. Peptide fragmentation
The peptide is protonated in positive mode of ion. The proton is located initially at the N-
terminus or making basic side chain residue, as there is internal solvation as it moves along the
backbone by breaking different sites that resulted in different fragmentation. The rules of
fragmentation can be explained well depending on the three different backbones bonds which
can be broken to form peptide amide bond (CO-NH), alkyl carbonyl (CHR-CO) and aminoalkyl
bond (NH-CHR).
The backbone cleaves six different sequence ion types. The fragment ions have charged
N-terminal that are grouped as a, b and c and the C-terminal charged ions are categorised as x, y
and z (Schymanski et al. 2014). The sequence ions a, b and y ions are the collective types of ion
in the low-energy category of CID (collision-induced dissociation) mass spectrometers as the
peptide amide bond causes the loss of CO from b-ions.
The fragment ions present in MS/MS spectrum depends on different factors that comprise
primary sequence, internal energy and the way in is introduced and state of charge. Fragments
detected carries at least one charge.
2.1.3 Side chain effects
The globular protein structure consists of hydrophobic side chains. The energy transferal
of side chains ranging from solvent accessible to solvent-inaccessible is the major reason for the
folding of the globular protein the aqueous state. The large hydrophobic buried side chains