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Samenvatting Protein technology and proteome analysis

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Summary of Protein technology, including the “advanced” section, based on notes, slides, lesson recordings, youtube videos and information from the specified book.

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  • Hoofdstukken 5, 15, 21 en 22
  • November 9, 2024
  • 34
  • 2023/2024
  • Summary
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1 Protein technology and proteome analysis

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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