Samenvatting Protein technology and proteome analysis
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Course
Protein technology and proteome analysis
Institution
Universiteit Antwerpen (UA)
Book
Principles and Techniques of Biochemistry and Molecular Biology
Summary of Protein technology, including the “advanced” section, based on notes, slides, lesson recordings, youtube videos and information from the specified book.
Introduction
Proteomics = a part of life sciences proteins are molecules consisting of AA proteome = set of all
proteins in a cell proteomics = study of the proteome.
Understanding the dynamic complexity by integrating an image of all aspects of proteins:
mRNA and protein profiles and their change over time
State and properties of proteins post-translational modifications, cellular localisation, alternative
splicing proteolytic degradation, oligomeric states, …
Proteome = complex one gene can give rise to a large amount of translational products prediction of
the exact sequence, structure or modifications is hard need of analytical techniques with high precision
and sensitivity knowing the proteoforms (= different forms of the same protein) for understanding disease
etiology.
Protein interference problem = difficult to know which proteins and/or proteoforms were in a complex
sample when digested complex sample can be
studied through different pathways:
Leaving the proteins intact: gel-based separation,
affinity purification with Ab, multiplex chips and
arrays, Edman degradation or LC-MS/MS
Working with digested proteins: liquid
chromatography or MS based identification
Intact proteins = you can retain the proteoform info
with digestion proteoform info is lost!
Identifying isoforms: peptides that are shared only give
information about a protein group/cloud unique peptides give information of the presence of the protein
isoform.
Sample preparation
= preparing a complex or simple protein sample for MS or MS/MS peptide mixture = end goal
Bottom-up approach = used 90% of the time digestion with trypsin
Top-down approach = electrospray ionization + fragmentation
Define your Know what the point is of the technique you will use
research question
Proteins have more AA diversity protein diversity:
diversity hydrophobic core and hydrophilic mantel in cytosolic proteins
membrane associated proteins have hydrophobic TM part and loose parts
are hydrophilic
protein complexes, …
Sample collection Some proteins are stable, others are lable dynamic properties use a fresh
sample!
Biobank sample is possible too fixed in formaldehyde proteins are
already linked together
Serum or plasma
Cell lines
Patient samples resection sample will be stabilized by freezing in liquid
nitrogen
,2 Protein technology and proteome analysis
Degradation has to be minimized stabilizing as soon as possible
using inhibitors:
o protease inhibitor
o phosphatase inhibitor
denaturation:
o chaotropic agents
o detergents
temperature:
o cold = reversible, heat ≠ reversible
adjusting pH
Protein extraction- Most of the proteins are present in the cell releasing by disruption of the cell
solubilisation membrane:
difference in samples
o organisms with a cell wall are more difficult to disrupt
o difference between tissues brain is easier than muscle
buffer used is important e.g. RIPA or NP40
o non-denaturising = proteins will still be active
o denaturising = proteins are inactivated
Methods of disruption
Mechanical sonication
bead beating
mixing
freeze thawing
Non-mechanical detergents
chaotropic agents
Soft osmotic shock
most detergents = breaking up the lipid membrane
enzymatic digestion lysozyme degrades peptidoglycan
dounce homogenizer opening cells based on shear
stress minimal heating + can keep organelles intact
Harsh blender or tissue chopper
(can create cryo-grinding with pestle and mortar in liquid N2
artefacts) bead beater = samples in vail with metallic beads
machine will shake, causing the beads to disrupt the
membrane for frozen or dry tissues
sonication shock waves to disrupt the tissues cooling
sample in between as heat is created
Solubilisation
Chaotropic agent Detergents/surfactant Commented [TN1]: Need to get rid of detergent if
disruption of non-convalent bonds hydrophobic region binds to hydrobic you want to digest the protein for MS → can
region of protein/membrane interfere with analysis → removal by:
disordering of water leads to
Dialysis
solubilisation of hydrophobic molecules hydrophilic region will form a bond Gel filtration chromatography
o Urea (7-8M) for 2D-PAGE with the solution Mass cut-off filter
efficient H-bond disruption formation of micel Protein precipitation (e.g. acetone)
o Thio-urea (2M) efficient Denaturing Non-denaturing Commented [TN2R1]: Also if you use a high amoun
hydrophobic disruption good of chaotropic agents!
o SDS = anionic o Non-ionic
for membrane proteins disrupts break lipid-lipid
o Guanidinium chloride (6M) membrane, PPI and lipid-
efficient H-bond and and protein protein bond
hydrophobic disruption activity (e.g. Tween)
o Zwitterionic =
no net charge
(e.g. CHAPS)
,3 Protein technology and proteome analysis
Protein separation After protein extraction you end up with sample of proteins + remnants of the cell:
or purification subcellular fractionation sucrose gradient or with differential
centrifugation
enrichment of certain organelles can be done too
Purification of proteins
acetone precipitation = purification of proteins after solubilisation
gel filtration = buffer with porous beads smaller molecules will pass
through porous bead = take a longer route/time than larger molecules
other filtration methods
SDS-PAGE
Reduction and Reducing the disulphide bridges (between Cys residues) reducing agent
alkylation o β-mercaptoethanol ionizes at high pH affects IEF in 2D-E
o Dithiothreitol (DTT) ionizes at high pH too, but needed in much lower
conc. can be used for IEF
After reduction the thiol groups are free highly active
o Alkylation = modification of the thiol group
o Iooacetic acid or iodoacetamide can be used to alkylated the thiol groups
aggressive chemicals: can have an effect on the mass effect on MS
Depletion of high e.g. albumin is highly abundant in the plasma can overshadow the proteins that
abundant proteins are low abundant depletion with antibodies = immunodepleting
Digestion Trypsin is used for digestion: Commented [TN3]: Labels can be added for
Peptides of 10-20 AA = optimal for LC-MS quantitative proteomics -> usually label at primary
Cleaves after lysine or arginine = AA that can be easily ionized amines (like Lys) -> done after digestion
Digestion overnight trypsin = robust can tolerate high T, high [urea],…
Other enzymes like chymotrypsin (cuts after AA that are aromatic or hydrophobic)
or GluC (cuts after Glu) can be used
Peptide purification C18 solid phase extraction = purification of peptides
or selection Separation based on hydrophobicity
C4/8 are used for larger peptides or proteins
Strong cation exchange tryptic peptides are usually positively charged
Prefractionation Highly complex samples simplifying the peptide sample before LC-MS
Amino-acid analysis
= determination of AA composition/content of a protein, peptides and pharmaceutical preparations
identification of exact primary AA sequence. Early on this was done with chemical and enzymatic
approaches currently done with mass spectrometry.
Usage For calibration of LC-MS isotopically labelled internal standard.
Estimation of contaminating proteins in DNA or other sample
Measurement of free AA in fluids/media e.g. PKU : phenylketonuria = disease
where Phe can’t be diverted into Tyrosine high amount of Phe can be lethal
tracing amount of Phe in food
o Food: identification of micronucrients, proteins, carbohydrates and vitamins
cryo-grinding, chopping or solvent extraction
o Physiological sample technique depends on kind
o Proteins and peptides: characterisation
AAA of proteins
and peptides Breakage of the peptide bond needs to be chemically stimulated hydrolysis of
liquid sample of gaseous sample separation of formed AA with cation exchange or
RP-HPLC
, 4 Protein technology and proteome analysis
Hydrolysis
Liquid phase hydrolysis Gas phase hydrolysis
Proteins are exposed to high concentration of HCL and high temperature some AA
will be degraded hydrolysis effects the AA:
Valine and isoleucine are largely resistant
Threonine and serine slowly degrade
Methionine will become partially oxidized
Asparagine and glutamine will be converted into their acidic forms
Tryptophan and cysteine will be fully degraded
corrections of these modifications need to be made done by Moore and Stein
Method
Separation
RP-HPLC – pre-column derivatization Cation exchange – post-column
derivatization
= most sensitive method Cation exchange = separation of AA
derivatization of the AA is done with phenyl based on charge and
isothiocyanate (PITC = Edman’s reagent) or o- hydrophobicity
phthalaldehyde (OPA) column is negatively charged
OPA with sulphonated polysterene
particles
AA separation: + AA will bind to
column and – AA will flow through
AA have certain elution profile
elution is realised by increasing pH =
When complexed with the AA, the less charge absorbance peak is
molecule becomes fluorescent = directly correlated w/ concentration
isoindole product enables sensitive of the sample
Commented [TN4]:
detection (ex: 348nm, em: 450nm)
Mecaptopropionin acid = SH donator Derivatization is done with ninhydrin
But there is no detection of AA that are of OPA:
secondary amines (Proline)
Ninhydrin
isoindole product = unstable, rapid
performance of RP-HPLC is needed
detector will identify OPA-AA
sensitivity of 50 fmol – 1 pmol
PITC
PITC + AA = PIT (phenylthiocarbamyl)
derivate can be detected at 254nm Interaction with AA gives a
derivates are more stable purple product (570nm)
less sensitive: 20 – 500 pmol Interaction with proline
RP-HPLC = separation based on hydrophobicity (imino acid) gives a yellow Commented [TN5]:
side chain will determine the elution time product (440nm)
C18 column used
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