2.1.1 the physical separation features of LC

2.1.1 Introduction to LC-MS

Before the discovery of
LC-MS, Gas Chromatography-Mass Spectrophotometry (GC-MS) was first introduced
by A.T. James and A.J.P Martin in 1952 while they were trying to create a
combined technique of separation and mass analysis (James & Martin, 1952). The first
development of LC-MS was initiated only in the early 1970s by V.L. Tal’roze.
They managed to couple liquid chromatography (LC) and mass spectrophotometry
(MS) by creating the capillary inlet interface which involved using capillaries
to connect LC columns and MS ion sources (Tal’roze,
et al., 1978).
LC-MS is an analytical chemistry method used in disease biomarkers discovery.
It combines the physical separation features of LC and the mass analysis
feature of MS. Using MS together with chromatographic techniques have always
been desirable due to the high sensitivity and specificity MS offers (James, 2009).

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2.1.2 The functions of LC-MS Electrospray Ionisation (ESI)

The ESI interface for
LC-MS was developed by Fenn in 1988 (Fenn, et al., 1989). This interface is used for the
analysis of moderately polar molecules such as metabolites and peptides. The
eluate from the LC column is first pumped through a metal capillary with
electric potential of 3 to 5 kilovolts (kV). This eluate is then nebulised at
the tip of the capillary and charged droplets are formed. The heat created by
the electric potential rapidly evaporates these charged droplets in the
presence of dry nitrogen. These ionised analytes are transferred into a high
vacuum chamber of the MS and flow through small holes by the aid of focusing
voltage, where positively charged and negatively charged ions are then detected
(James, 2009). Atmospheric Pressure Chemical Ionisation (APCI)

The APCI interfaced for
LC-MS was developed by Horning in 1973 (Horning, et al., 1973). In 1986, Henion
improved the interface (Willfried, 2006) and its commercial application only
began in the early 1990s. This interface is used for the analysis of small,
neutral, relatively non-polar and thermally stable molecules such as lipids and
steroids, which do not ionise properly in the ESI interface. Furthermore, APCI
can be used with buffering agents containing mobile phases. Similar to ESI, the
eluate from the LC column is pumped through a capillary and nebulised at the
tip, where a corona discharge occurs. There is ionising gas surrounding the
interface which later reacts with the analytes, transferring their charge.
These ionised analytes will then undergo mass analysis in the high vacuum
region of MS (James, 2009). Atmospheric Pressure Photo-Ionisation (APPI)

The APPI interface for
LC-MS was developed by Bruins and Syage in 2000 (Robb, et al., 2000). It is used for the analysis of neutral
compounds that also cannot be ionised by ESI (James, 2009).
Similar to APCI, eluates will be pumped through a capillary and nebulised at
the tip. However, instead of a corona discharge, ionisation is achieved using
photons from a discharge lamp. There are two modes in APPI, the direct-APPI
mode and dopant-APPI mode. The direct-APPI mode analytes are singly charged by
absorption of a photon and ejection of an electron. On the other hand,
dopant-APPI mode uses an ionisable compound (Dopant) in the mobile phase or
nebulising gas to promote charge-exchange reactions between dopant ions and
analyte. The charged analytes will then undergo mass analysis in the high
vacuum region (Willfried, 2006).


2.1.2 Uses of LC-MS in proteomics in discovery of disease

LC-MS is used in
proteomics to identify components in a complex mixture. The approach to using
LC-MS involves protease digestion, denaturation of protein using trypsin as a
protease, breaking down of tertiary structure using urea and finally the
modification of cysteine residues using iodoacetamide. After the cascade of
digestion, peptide mass fingerprinting or tandem MS is conducted to determine
the sequence of individual peptides (Vicki, et al., 2005). From there, the
masses of unknown proteins will be compared to a database of known theoretical
protein masses. Subsequently, the unknown protein may be identified. However,
when identifying a complex mixture with thousands of proteins, such as human
serum, an additional step of sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) has to be conducted for higher specificity of
identification (Sudhakar, et al., 2016).