PHA6112 LAB_Proteomics development.

• Biologically-active (bioactive) peptides

• peptide fragments, whose pharmacological
activities are greatly
Additional notes:
influenced by their length and amino acid
composition
PEPTIDE SEQUENCING : OUTLINE
• usually have 2-20 amino acid residues
• Introduction
(Gonzalez-Ortega et al., 2015)
• Biological sources of proteins
• Protein sample preparation
INTRODUCTION : Peptides as Drugs
• Protein digestion
• Peptide separation/isolation
• Peptide sequencing

Additional notes:
Proteomics is the large-scale study of
proteomes
● A proteome is a complete set of
proteins produced in an organism,
system, or biological context.
WHY DO WE NEED TO STUDY IT? STRENGTHS
● Sequence proteins ● Good efficacy, safety and tolerability
○ Following the identification of ● High selectivity and potency
proteins, you may perform
● Predictable metabolism
bioassays, safety tests, etc.
● Determine the function of protein ● Shorter time to market
● If there is a pharmacological effect WEAKNESSES
then it will be synthesized and mass ● Chemically and Physically unstable
produced. OPPORTUNITIES

PROTEINS VS. PEPTIDES THREATS

○ Proteins are polypeptides
● Immunogenicity
○ Proteins are larger compared to
polypeptides ● New advancements in Genomics,
○ Polypeptides are fragments of Proteins proteonimics and personalized medicine
Additional notes:
INTRODUCTION ● They are very prone to disintegration,
• The vast number of biologically active peptides especially if taken through the oral
existing in route.
nature (plants, animals, microorganisms) ○ They are denatured in an
represent one of the environment with extremely
most promising new sources for drug discovery
and
 low pH, such as in the
stomach.
○ The intestines also contain
enzymes which could
hydrolyze proteins.
● Proteins are commonly found in
vaccine formulation.
○ Vaccines which contain
proteins are used to target
certain microorganisms with
proteinaceous structure.
have an effect or drug activity.
● At times, however, data from the
database do not match actual test
results when performed in live
subjects.
WHAT IS THE FACTOR THAT MAY AFFECT THE
RESULTS FROM LIVING ORGANISMS FROM
RESULTS FROM COMPUTERS?
- Metabolism

Additional notes:
Additional notes:
NMR:Nuclear Magnetic Resonance, an
Empirical Approach:
instrument allows the molecular structure of a
● Conventional method
material to be analyzed by observing and
● Through observations and
measuring the interaction of nuclear spins
experimentations
when placed in a powerful magnetic field.
● Structure-activity data deposited to
database

Bioinformatics Approach:
● New method
● Computer-generated (use of AI)
● Since empirical approach has
produced numerous data, database
mining can be performed.
● Database of receptors, etc.
● If interaction occurs, then peptide may
I. BIOLOGICAL SOURCES OF PROTEINS
• Animals
• “Zootherapeutic peptides”
• animal secretions, venom, dried animal parts,
Additional notes:
etc. ● Precipitation occurs when charge and
• Peptides from spider venom hydration are disturbed.

• Plants/Algae
Protein Precipitation Techniques:
• “Botanical peptides”
• Salting out
• herbs, roots, leaves, etc.
• Isoelectric Precipitation
• Food/food products
• Precipitation by Organic Solvents
• Fruits and vegetables, albumin, cheese, milk etc.
• Precipitation by Heavy Metal Ions
• Fungi
• Precipitation by Alkaloidal Reagents
• mushrooms, yeasts, etc.
• Bacteria
• SALTING OUT
• bacterial peptides (i.e. Vancomycin, Gramicidin
• When a neutral salt, such as ammonium
A,B and C)
sulfate or sodium sulfate is added to protein
• Cell culture
solution, the shell of hydration is removed and
• process by which cells are grown and
the protein is precipitated.
maintained under carefully controlled conditions
• As a general rule, higher the molecular weight of
outside of a living organism
a protein, the salt required for precipitation is
lesser.
Additional notes: Basically, a competition between protein and salt
● Fungi may be poisonous since it came Higher MW=lesser Salt required
from the wild. Myoglobin: used for salting out

● Vancomycin is used to treat infections

caused by bacteria. Additional notes:
● Ammonium sulfate and sodium sulfate
have high affinity for water. They tend
II. PROTEIN SAMPLE PREPARATION
to compete with proteins, which
• Purification of enzymes and other proteins results in protein precipitation.
usually start with precipitating them from
solution.
• The stability of proteins in solution will depend
•ISOELECTRIC PRECIPITATION
mainly on the charge and hydration.
• Proteins are least soluble at their isoelectric pH.
• Polar groups of the proteins (NH2, COOH, OH
• Some proteins are precipitated immediately
groups) tend to attract water molecules around
when adjusted to their isoelectric pH. The best
them to produce a shell of hydration. Any factor,
example is casein which forms a occulent
which neutralizes the charge or removes water
precipitate at pH 4.6 and redissolves in highly
of hydration will therefore cause precipitation of
acidic or alkaline solutions.
proteins.
ipH-> neutral->no charge-> nonpolar->non-ionized
-> insoluble-> precipitates
• PRECIPITATION BY ORGANIC SOLVENTS
• When an organic solvent is added to the protein
solution, water molecules available for proteins
are reduced, and precipitation occurs.
• Organic solvents reduce the dielectric constant
of the medium which also favors protein
precipitation
Alcohol:Antiseptic/ disinfectant
Separatory funnels
Additional notes:
● Like neutral salts, organic solvents
also compete with proteins.
● The capacity of organic solvents to
precipitate proteins make alcohol a
good anitseptic.
● Fractionation method is based on
polarity.
● Carbon tetrachloride and chloroform
(semi-polar) are used to extract
proteins.
• PRECIPITATION BY HEAVY METAL IONS
• In alkaline medium, proteins have net negative
charge, or are anions. To such a solution, if salts
of heavy metals are added,
positively charged metal ions can complex with
protein molecules
and metal proteinates are precipitated.
Additional notes:
p(-)+ Metals(+)=>Precipitate
Albumin(-) + Pb(+) = Pb albuminate
● Since albumin can form complex with
lead, raw egg (which contains albumin)
can be used as an antidote for
firecracker poisoning (primarily due to
lead). The complex formed will cancel
the effect of the poison.
PRECIPITATION BY ALKALOIDAL REAGENTS
• Tungstic acid, phosphotungstic acid, trichloro
acetic acid, picric acid, sulfosalicylic acid and
tannic acid are powerful protein precipitating
agents. These acids lower the pH of medium,
when proteins carry net positive charges. These
protein cations are complexed with negatively
charged ions to form protein tungstate,
protein-picrate, etc., and thick occulent
precipitate is formed.
p(+) + A(-) => Precipitation
III. PROTEIN DIGESTION
• Peptide fragments can
be produced from precursor/parent proteins
through protein digestion or hydrolysis.
• Hydrolysate – product of digestion/hydrolysis
• most common way to produce peptide
fragments from whole proteins.
• use proteases/peptidases
IN VITRO ENZYMATIC HYDROLYSIS
• Peptidases
• exopeptidases – cleaves external peptide
bonds
• Aminopeptidase – sequentially cleave peptide
bonds beginning at the n-terminal end
• Carboxypeptidase – sequentially cleave peptide
bonds beginning at the c-terminal
end
• endopeptidases – cleaves internal peptide
bonds
• Trypsin – cleaves peptide bonds at the carboxyl
end of 2 strongly basic aas: arg & lys
• Chymotrypsin – cleaves peptide bonds at the
carboxyl end of 3 aromatic aas: phe,tyr, & trp; leu
• Pepsin – cleaves peptide bonds at the amino
end of 3 aromatic aas: phe, tyr, & trp;ileu

N-AA1-AA2-AA3-C

PEPTIDASES
Additional notes:
● Proteins are hydrolyzed since peptide EXOPEPTIDASES ENDOPEPTIDASES
fragments are usually the ones
exhibiting activity. DEFINITION:cleaves DEFINITION:cleaves
external peptide internal peptide
bonds bonds
Protein Digestion Techniques:
• In vitro enzymatic hydrolysis
AMINOPEPTIDASE TRYPSIN
• Hydrolysis during microbial fermentation
sequentially cleave cleaves peptide bonds
• Hydrolysis during gastrointestinal peptide bonds at the carboxyl end of
digestion(HCl,pepsin) beginning at the 2 strongly basic AAs:
• Acid hydrolysis n-terminal end Arg(R) & Lys(K)
• Alkaline hydrolysis

CARBOXYPEPTIDASE CHYMOTRYPSIN
IN VITRO ENZYMATIC HYDROLYSIS sequentially cleave cleaves peptide bonds
 and Streptococci) used in the manufacture of
peptide bonds at the carboxyl end of
beginning at the 3 aromatic AAs: fermented dairy products.
c-terminal Phe(F),Tyr(Y), &
end Trp(W); leu(L) • HYDROLYSIS DURING GASTROINTESTINAL
DIGESTION
• It is now well-known that physiologically active
PEPSIN
cleaves peptide bonds peptides are produced from several food proteins
at the amino end of 3 during gastrointestinal digestion in humans.
aromatic AAs:
Phe(F), Tyr(Y), & HYDROLYSIS DURING GASTROINTESTINAL
Trp(W);ileu(I) DIGESTION
• Dietary proteins and peptides are subjected to
G-H-R-W-I-H-G hydrolysis
during the various stages namely ingestion,
•HYDROLYSIS DURING MICROBIAL digestion and
FERMENTATION absorption. Ingested proteins are first hydrolyzed
• Fermentation is an efficient method and the by proteinases such as pepsin, trypsin and
cheapest way to generate bioactive peptides and chymotrypsin in the gastrointestinal tract to
hydrolyzed proteins. produce peptides of various sizes.
• For example soybean protein possesses These peptides are further digested by brush
numerous bioactive peptides, which are inactive border peptidases at the surface of intestinal
within the sequences of parent proteins but can epithelial cells to produce amino acids, while
be released through proteolytic hydrolysis during some oligopeptides remain intact.
fermentation.
Additional notes:
● Endopeptidases = pepsin, trypsin,
Additional notes: chymotrypsin
● S.aureus protease->D and E
• ACID HYDROLYSIS
•HYDROLYSIS DURING MICROBIAL • Heating (110ºC) + HCl (also: cyanogen Br)
FERMENTATION • Destroys Trp
• Many dairy starter cultures are highly (It should not be utilized since in destroys a
proteolytic. certain AA)
• Lactic acid bacteria (LAB) utilize milk proteins,
principally
Additional notes:
the caseins, as their prime source of essential ● Destruction of W will hinder accurate
and growth stimulating amino acids. Hence, determination of protein sequence.
bioactive peptides can be generated by starter
and non-starter bacteria (Lactobacilli, Lactococci
• ALKALINE HYDROLYSIS
• Heating + NaOH • ONLY THE C- TERMINAL AMINO ACID IS
• Does not damage Trp but destroys a lot more SPARED
AAs
V. PEPTIDE SEPARATION/ISOLATION
• Protein Separation Techniques:
• Chromatographic separation
IV. IDENTIFICATION OF PRODUCTS OF • Electrophoresis
HYDROLYSIS
• N-TERMINAL DETERMINATION SOURCE
• EDMAN’S METHOD PREP
• RGT: PHENYLISOTHIOCYANATE (PITC/ PH – N DIGEST
= C = S) IDENTIFICATION
• Cleaves the Carboxyl side of the N-Terminal SEPARATION
Amino Acid which produces a product, SEQUENCING
N-terminal amino acid +
Phenylthiohydantoin(PTH) CHROMATOGRAPHY
• Method used to separate and identify
components of
Additional notes:
● Action is similar to aminopeptidase a mixture based on differential affinities of the
solute
between 2 phases.
• FREDERICK SANGER’s METHOD (SANGER”S
• Stationary phase (SP)
METHOD)
• Fixed phase
• RGT: 1-FLUORO-2,4-DINITROBENZENE(DNFB)
• Thin/internal phase
• Mobile phase (MP)
Additional notes: • Fluid which moves over the surface of the
● Sanger was the first to identify the stationary phase
primary structure of insulin. • Bulk or external phase

ENDOPEPTIDASE • Classification
• CYANOGEN BROMIDE METHOD • Normal Phase
• Cleaves the carboxyl side of METHIONINE • SP: Polar
• Ex. BIOCHEMISTRY +Cyanogen Bromide • MP: Non-polar
• BIOCHEM + ISTRY • Reverse Phase
• SP: Non-polar
• C-TERMINAL DETERMINATION • MP: Polar
• HYDRAZINE METHOD
• HYDRAZINE REACTS WITH ALL AAs WHOSE Stationary phase
CARBOXYL GROUP IS BOUND IN PEPTIDE TLC PLATE(Silica)
LINKAGE, CREATING AMINO ACYL HYDRAZIDES - Polar
 - Mobile phase:BAW(Non-polar)
- Kapag mas malapit sa point of origin,
mas polar
• Size Exclusion Chromatography
̶Molecules are separated based on their
molecular size by
passage through molecular sieves (eg. gel)
• Ion-exchange chromatography
̶Separation of ions based on their charge using
ion exchange resin
• Affinity Chromatography
̶Analysis of antigens and antibodies
̶Stationary Phase: packed column
̶Normal phase (polar): silica
̶Reverse phase (non-polar): ODS (octadesylsilane;
C18)
̶Mobile Phase: solvent
̶Isocratic: MP w/ constant composition (1
reservoir)
̶Continuous: MP w/ changing composition (≥2
reservoirs)
Additional notes:
● Continuous involves change in ratio of
mobile phase constituents.

Chromatographic Techniques in Protein

Separation
• HIGH PRESSURE LIQUID CHROMATOGRAPHY
PROTEINS
(HPLC)
• COLUMN CHROMATOGRAPHY
̶ High pressure = faster separation
̶ Stationary Phase: silica gel(Polar) beads
̶ Chromatogram: peak intensity vs TR
placed in a cylindrical tube
constricted on end (column)
̶ Mobile Phase: solvent Additional notes:
̶ Basis of protein separation: ● In some studies, HPLC is simply
Acidity/alkalinity referred to as LC.
Polarity ● Results are presented in a graph,
Affinity where TR = x value and INTENSITY = y
value.
● Aside from a container for waste, the
machine’s detector has a collector
where desired component is collected.

V. PEPTIDE SEPARATION/ISOLATION
PROTEINS
• SIZE-EXCLUSION/ GEL-FILTRATION
CHROMATOGRAPHY
̶ Employs porous beads
̶ Separates proteins using stokes radii (measure
Serological pipette will be used in the lab
of effective

• HIGH PRESSURE LIQUID CHROMATOGRAPHY

(HPLC)
vol. Occupied by protein as it rapidly tumbles in
free sol’n) à subsequent proteins elute in order of
decreasing size
̶ Basis of protein separation: molecular mass &
shape
Molecules with larger size will be eluted first
Additional notes:
● Gels form pores after they swell, which
takes place once they are mixed with a
buffer.
● Addition of buffer with a different
concentration will cause the gel to
shrink, resulting in the elution of small
molecules.
Electrophoresis
SODIUM DODEACYL SULFATE-
POLYACRYLAMIDE GEL
ELECTROPHORESIS(SDS-PAGE)
̶ Acrylamide: polymerized & cross-linked as a
porous matrix
̶ SDS: binds to proteins (1 molecule of SDS: 2
peptide bonds)
̶ 2-mercaptoethanol or dithiothreitol: reduce &
break SH bonds
̶ Coomassie blue: visualizing dye
̶ Basis of protein separation: molecular mass
(larger complexes encounter greater resistance)
High MW=Slow rate
Low MW= Fast rate
Additional notes:
● Buffer will facilitate separation based
on molecular weight.
● Proteins develop affinity for the gel.
Why are peptides, and not proteins,
sequenced?
• Proteins can be difficult to handle and might
not all be soluble under the same conditions;
• The sensitivity of the mass spectrometer for
proteins is much lower than for peptides; and
• The mass spectrometer is most efficient at
obtaining sequence information from peptides
that are up to ~20 residues long, rather than
from whole proteins.
Additional notes:
● Proteins are too large for sequencing.
● Peptides are also the ones responsible
for activity.
VI. PEPTIDE SEQUENCING
ESI:Perfume-like
MALDI: Sensitive to high temperature
FAB: Applied to volatilize large, intact
macromolecules
Additional notes:
● MALDI causes peptide excitation,
allowing for their sequencing.
○ However, since it uses a laser,
heat is involved, making
denaturation possible.

Additional notes:
● Bioactive compounds exhibit high
peaks.
○ Lower peaks = noise

SINGLE QUADRUPOLE MS: <4000 Da

Low MW: faster rate

Additional notes:
● MALDI-TOF
○ Dependent on time of flight
○ During excitement, molecules
with low MW reach the mass
analyser faster since they
have a shorter TOF.

Protein Sequencing and Identification by Mass

Spectrometry

Additional notes:
● Goal: To determine the molar mass
Breaking Protein into Peptides and Overlapping Technique
Peptides into Fragment Ions
• Proteases, e.g. trypsin, break protein into
peptides.
• A Tandem Mass Spectrometer further breaks
the
peptides down into fragment ions and measures
the
mass of each piece. Kung sino yung magrereact sa reagent, siya yung
• Mass Spectrometer accelerates the fragmented nasa end terminal
ions;
heavier ions accelerate slower than lighter ones.
• Mass Spectrometer measure mass/charge ratio
of an ion.(m/z)

Additional notes:
● Accelerate = TOF
● Mass/charge refers to fragment ions.

R,S,D,G,W,M,A,F,C
G-S-F|D-M-W|C-R-A
ANSWER:PRO- GLY- TRP- ARG- ALA

ANSWER: CYS-TRY-ILE-GLN-ASN-PRO-LEU-GLY