LC-MS combines liquid chromatography separation with mass spectrometry detection. It is useful for bioactivity screening, proteomics, and other applications. Key aspects of LC-MS include the HPLC separation, ionization sources like ESI and APCI, mass analyzers like quadrupole and time-of-flight, and the ability to identify proteins and peptides from complex mixtures. Proteomics uses these techniques to study protein expression, structure, function, and interactions on a large scale.

1. LC-MS IN BIOACTIVITY
SCREENING AND
PROTEOMICS
By
S.Nithya
Pharmaceutical Analysis
Second semester

2. LIQUID CHROMATOGRAPHY-MASS
SPECTROMETRY
 Liquid chromatography – mass spectrometry (LC/MS) is a
technique that uses liquid chromatography (or HPLC) with
the mass spectrometry.
 It is an analytical chemistry technique that combines the
physical separation capabilities of liquid chromatography
with the mass analysis capabilities of mass spectrometry.

3. Basic Principle of LC/MS:
LC/MS combines the separating power of High
Performance Liquid Chromatography (HPLC), with the
detection power of Mass Spectrometry.
1. High Performance Liquid Chromatography (HPLC)
Liquid chromatography involves two main phases:
Mobile phase: Liquid (solvents i.e. Ethanol,
Acetonitrile)
Stationary phase: Column packed with very small
particles.
2. Mass Spectrometry:
Analytical technique that measures the mass –to-
charge ratio of charged particles.

4. Theory of LC/MS
 HPLC is a method for separating a complex mixture in
to its components.
 High sensitivity of mass spectroscopy provides the
information for identification of compounds or structural
elucidation of compounds.
 Combination of these two techniques in LC-MS.
 As the metabolites appear from the end of the column
they enter the mass detector, where the solvent is
removed and the metabolites are ionized.

5. PROBLEMS IN COMBINING HPLC AND MS
HPLC MS
 Liquid phase operation
 25-50 deg. C
 No mass range limitations
 Inorganic buffers
 1 ml/min eluent flow is
equivalent to 500 ml/min
of gas
 Vacuum operation
 200-300 deg .C
 up to 4000 Da for
quadrapole MS
 Requires volatile buffers
 Accept 10 ml/min gas
flow

6. High Pressure Liquid Chromatography
 Liquid chromatography generally utilizes very small
particles packed and operating at relatively high
pressure, and is referred to as high performance liquid
chromatography (HPLC).
 Modern LC/MS methods use HPLC instrumentation,
essentially exclusively, for sample introduction.
 In HPLC, the sample is forced by a liquid at high
pressure ( the mobile phase) through a column that is
packed with a stationary phase generally composed of
irregularly or spherically shaped particles chosen or
derivatized to accomplish particular types of separation.
 RP-LC is most often used as the means to introduce
samples into the MS, in LC-MS instrumentation.

7. MASS SPECTROMETRY
 Mass spectrometry (MS) is an analytical technique that
measures the mass-to-charge ratio of charged particles.
 MS works by ionizing chemical compounds to generate
charged molecules or molecule fragments and measuring
their mass-to-charge ratios.
 In a typical MS procedure, a sample is loaded onto the
instrument and undergoes vaporization.
 The components of the sample are ionized by one of a
variety of methods ( e.g., by impacting them with an
electron beam), Which results in the formation of
charged particles (ions).
 The ions are separated according to their mass-to-charge
ratio in an analyzer by electromagnetic fields.

8. INSTRUMENTATION OF LC/MS
 1) HPLC constitutes the lc part:
 Solvent system (mobile phase)
 Pumps
 Mixer
 Injector
 Column
 2) Mass spectrometer
 A) Ion sources
 Electrospray ionization
 Atmospheric pressure chemical ionization
 Atmospheric pressure photoionization
 B) Mass Analyzer
 Quadrupole
 Time of flight
 Ion trap
 Fourier transform-ion cyclotron resonance

10. MOBILE PHASE
The mobile phase is the solvent that moves the solute
through out column.
General requirements:-
1) Low cost, UV transparency, high purity.
2) Low viscosity, low toxicity, non flammability.
3) Non corrosive to LC system component.
Solvent strength and selectivity:-
It is the ability of solvent to elute solutes from a
column.

11. COLUMN
The use of di-functional or tri-functional silanes to
create bonded groups with two or three attachment
points leading to phases with higher stability in low or
higher PH and lower bleed for LCMS
Most widely used columns for LC-MS are:-
1) Fast LC column.
the use of short column. (15-50mm)
2) Micro LC column.
the use of large column. (20-150mm)

12. SAMPLE PREPARATION
Sample preparation generally consists of
concentrating the analyte and removing compounds that
can cause background ions or suppress ionization.
Examples of sample preparation include:
 On-column concentration to increase analyte
concentration.
 Desalting to reduce the sodium and potassium adduct
formation that commonly occurs in electrospray
 Filtration to separate a low molecular-weight drug from
proteins in plasma, milk, or tissue.

13. ION SOURCES
Ion sources :
Electrospray ionization (ESI)
Atmospheric pressure chemical ionization (APCI)
Atmospheric pressure photoionization (APPI)
 Electrospray ionization (ESI)
 Process
API-ES is a process of ionization followed by
evaporation. It occurs in three basic steps:
1. Nebulization and charging
2. Desolvation and;
3. Ion evaporation

14. NEBULIZATION:
 The Hplc effluent is pumbed through a nebulizing needle
which is at ground potential.
 The spray goes through a semi-cylindrical electrode Which
is at a high potential.
 The potential difference between the needle and the
electrode produces a strong electrical field.
 This field charges the surface of the liquid and forms a
spray of charged droplets.
 There is a concentric flow of gas which assists in the
nebulization process.

16. DESOLVATION:
 The charged droplets are attracted toward the capillary
sampling orifice.
 There is counterflow of heated nitrogen gas which shrinks
the droplets and carries away the uncharged material.
IONIZATION:
 As the droplets shrink, they approach a point where the
electrostatic (coulombic) forces exceed the cohesive forces.
 This process continues until the analyte ions are ultimately
desorbed into the gas phase.
 These gas-phase ions pass through the capillary sampling
orifice into the low pressure region of the ion source and
the mass analyzer.

17. Atmospheric Pressure Chemical Ionization:
 Process:
APCL, a process of evaporation followd by ionization,
is complementary to API-ES.
NEBULIZATION AND DESOLVATION:
 APCI nebulization is similar to that in API-ES.
 However, APCI nebulization occurs in a hot (typically
250ºC-400ºC) vaporizer chamber.
 The heat rapidly evaporates the spray droplets, resulting in
gas-phase HPLC solvent and anlytes molecules

18. IONIZATION:
 The gas-phase solvent molecules are ionized by
the discharge from a corona needle.
 In APCI there is a charge transfer from the
ionized solvent reagent ions to the analyte
molecules in a way that is similar to chemical
ionization in GC/MS.
 These analyte ions then are transported through the
ion optics to the filter and detector.

19. ATMOSPHERIC PRESSURE PHOTOIONIZATION
 Atmospheric pressure photoionization (APPI) for LC/MS is
a relatively new technique.
 As in APCI, a vaporizer converts the LC eluent to the
gas phase.
 A discharge lamp generates photons in a narrow range of
ionization energies.
 The range of energies is carefully chosen to ionize as
many analyte molecules as possible while minimizing the
ionization of solvent molecules.
 The resulting ions pass through a capillary sampling
orifice into the mass analyzer.

21. MASS ANALYZERS
Mass analyzer is also called ion separator.
Mass analyzer is the heart of the mass spectrophotometer
that takes ionized masses and separates them based on
charge to mass ratios.
TYPES OF MASS ANALYZERS
 Quadrupole mass analyzer
 Time of flight analyzer (TOF)
 Quadrupole ion trap mass analyzer

22. QUADRUPOLE:
A quadrupole mass analyzer consists of four parallel rods
arranged in a square.
The analyte ions are directed down the center of the
square.
Voltages applied to the rods generate electromagnetic
fields.
These fields determine which mass-to-charge ratio of ions
can pass through the filter at a given time.
Quadrupole tend to be the simplest and least expensive
mass analyzers.

24.  Quadrupole mass analyzers can operate in two modes:
 Scanning (scan) mode.
 Selected ion monitering (SIM) mode
 Scan mode:-
The mass analyzer monitors a range of mass-to-charge ratios.
In mass analyzer monitors only a few mass-to-charge ratios.
 SIM mode:-
Significantly more sensitive than scan mode but provides
information about fewer ions.
Scan mode is typically used for qualitative analyzes or for
quantification when all analyte masses are not known in advance.
SIM mode is used for quantification and monitering of target
compounds.

25. TIME OF FLIGHT
 In a time of flight (TOF) mass analyzer, a uniform
electromagnetic force is applied to all ions at the same
time, causing them to accelerate down a flight tube.
 Lighter ions travel faster and arrive at the detector first,
so the mass-to-charge ratios of the ions are determined
by their arrived times.
 Time-of-flight mass analyzers have a wide mass range
and can be very accurate in their mass measurements.

27. ION TRAP
 An ion trap mass analyzer consists of a circular ring
electrode plus two end caps that together from a chamber.
 Ions entering the chamber are “trapped” there by
electromagnetic fields.
 Another field can be applied to selectivity eject ions from
the trap.
 Ion traps have the advantage of being able to perform
multiple stages of mass spectrometry without additional
mass analyzers

29. DETECTORS
 Once the ions have passed the mass analyzer they have
to be detected and transformed into a usable signal.
 The detector is an important element generating secondary
electrons, which are further amplified, or by inducing a
current (generated by moving charges).
 Ion detector fall into two main classes:
 Point detectors
 Array detectors

30.  POINT DETECTOR:
Ions are not spatially resolved and sequentially
impinge upon a detector situated at a single point
within the spectrometer geometry
 ARRAY DETECTOR:
Ions are spatially resolved and all ions arrive
simultaneously (or near simultaneously) and are recorded
along a plane using a bank of detector.

31. APPLICATION OF LC/MS
 Pharmaceutical Applications:
Rapid chromatography of benzodiazepines
Identification of bile acid metabolite
 Biochemical applications:
Rapid protein identification using capillary
LC/MS/MS and database searching.
 Clinical Applications:
High-senstivity detection of trimipramine and
thioridazine

32.  Food Applications:
Identification of aflatoxins in food
Determination of vitamin D3 in pourity feed
supplements
 Environmental Applications:
Detection of phenylurea herbicides
Detection of flow levels of carbaryl in food
 Forensic Applications:
Illegal substanes, toxic agents
Explosives

33. PROTEOMICS
 Proteome is a defines the complete set
of proteins expressed during a cell’s entire
lifetime.
 Proteomics is the study of the proteome;
it uses technologies ranging from genetic
analysis to mass spectrometry.
 Proteomics assesses
 Activities
 Modifications
Localization
Interactions of proteins in
complexes

34. ROLE OF PROTEOMICS
 To study the structure and function of protein
 To study the 3D structure of protein
 Study of qualitative and quantitative analysis of
proteins

35. PROTEOMICS OBJECTIVES
 Protein/peptide separation.
 Identification and characterization of resolved
proteins by MS.
 Data analysis and applications.

36. INTRODUCTION
 Proteome indicates the total proteins expressed
by a genome in a cell or tissue.
 With the development of proteomic techniques,
proteome analysis provides a fast, non – invasive
diagnostic tool for patients with various diseases.
 The advent of highly sensitive proteomic
technologies can identify proteins associated with
development of diseases well before any
clinically identifiable alteration.
 MS has a high resolving power and identifies
proteins with more accuracy.

37. TYPES OF PROTEOMICS
1. Structural Proteomics:- The ultimate aim of this
proteomics is to build a body of structural
information that will help predict the probable
structure and potential function for almost any
protein from knowledge of its coding sequence.
2. Functional proteomics:- It refers to the use of
proteomics techniques to analyze the characterization
of molecular protein-networks involved in a living
cell.
3. Expression proteomics:- It refers to the quantitative
study of protein expression between sample differing
by some variable.

38. PROTEIN STRUCTURE
 Primary structure:- is sequence
of specific amino acid in
polypeptide chain.
 Secondary structure:- the
primary polypeptide chain gets
properly folded in the form of
alpha-helix, beta pleated sheet,
random coils and turns.

39.  Tertiary structure:- Secondary
structure interact with each
other chemically to form the 3
dimensional shape of the
proteins.
 Quardinary structure:-
interaction between different
polypeptide unit.

40. EXPERIMENTAL WORK FLOW
Proper sample selection and sample storage
Sample pre- processing
Immuno depletion/protein concentration etc
Protein separation by 1D SDS-PAGE/ 2DE
or
Fractionation by LC
In gel trptic digestion/ in-solution digestion
Protein in identification LC-MS/MS
Protein identification (sequest & Mascot
Bioinformatic Analysis

41. PROCEDURE OF PROTEOMICS
o Separation of proteins
one dimensional electrophoresis
2D electrophoresis (modern)
Multi-dimensional HPLC (modern)
o Analysis of proteins
Mass Spectrometry (modern)
o Database utilization

42. FIVE STEPS OF PROTEOME
ANALYSIS
1. Sample collection, handling and storage.
2. Separation of individual proteins by 2-D electrophoresis.
3. Protein characterization.
4. Identification by mass spectrometry or other methods.
5. Storage, manipulation, and comparison of the data using
bioinformatics.

43. 2-D GEL
ELECTROPHORESIS
 2-D gel electrophoresis a method for the separation and
identification of proteins in a sample by displacement in 2
dimensions.
 First step is to separate based on charge or isoelectric
point, called isoelectri focusing.
 Then separate based on size (SDS-PAGE).

44.  Separation of proteins by PI value
 Soaking the gel in SDS solution and fitting it on an
SDS PA gel
 Separating the proteins by molecular mass with SDS
PAGE

45. MASS SPECTROMETRY
INTRODUCTION
 MS/ MS plays important role in protein identification
(fast and sensitive)
 Derivation of peptide sequence an important task in
proteomics
 Derivation without help from a protein database (“ de
novo sequencing “), especially important in identification
of unknown protein
3 Major part
 Source ionized the sample
 Analyzer separate the ions on m/z ratio
 Detector sees the ions and analyzed the
result

47. Basic steps
 Isolate cell or other protein source
 Lyses cells and isolate proteins
 Break up protein into smaller ( but still relatively large)
amino acid chains
 Separate chains (2D Gel, gas or liquid chromatography)
 Analyze separated protein parts by mass spectrometry
Mass Spectrum
 Proteins consist of 20 different types of a. a. With
different masses (except for one pair leu and lle)
 Different peptides produce different spectra
 Use the spectrum of a peptide to determine its
sequence

48. Protein Identification
1.Peptide mass fingerprinting
 Protein is cleave in smaller peptides
 Masses measured with MS
 These masses are then compared to known
protein
 Computer programs translate the known genome
of the organism into proteins
 Cut the proteins into peptides and calculate the
masses of peptides
 Compare the known and unknown protein

49. 2.Tandem MS
 Two Mass Specs, (MS1, MS2)
 A specific peak corresponding to a specific peptide
chain is identified and fragmented to from ions
 The ions are analyzed by MS2, and identified as amino
acids
 This way each selected piece of the whole protein can
be broken up and analyzed

50. Bottom up approach
 In this method by
using mass
spectrometer, entire
peptides of proteins
is determined
 Advantages
Small masses are
easier to be handling
Top down approach
 By using mass
spectrometer, entire
protein is determined
without solution
digestion
 Advantages
It provide the
complete covering of
protein

51.  Electro spray ionization (ESI)
• In this method high electric field is applied to
the tip of capillary, from which solution will
pass through and get the ions of interest.
• Ions said to be multiple ions
• Multiple charged ions measure the high mass
biopolymer

52.  Matrix assisted laser desorption/ ionization
(MALDI)
By pulses of laser light on the sample, ions of
interest formed.
large bio molecules can be determined and
synthetic polymer greater than 200,000 Dalton.
Advantages
• High speed
• Relative immunity to contaminants

53.  Time of flight
MALD with MS is called time of flight or
Tof.
This enable and fast molar masses
determination along with determined impurities
and sequencing repeated units.
 It is a method of mass spectrometry in which
an ion’s mass-to-charge ratio is determined via
a time measurement.

54. Application of proteomics
 Protein sample identification/confirmation
 Proteins sample purity determination
 Detection of post-translation modification
 Detection of amino acids substitution
 Mass fingerprint identification of proteins
 Nutrition Research
 To identify unknown protein of interest
 Quantify protein and peptide
 Protein Biomarker

55. THANK YOU