PROTEIN TECHNOLOGY AND PROTEOMICS
GENERAL INFORMATION
The lessons are recorded
, LESSON 1 – INTRODUCTION (PROF. VAN OSTADE)
Proteomics is the determination of the complete set of proteins that is present in a system, under specific circumstances:
• This system may be:
o Protein complex
o Subcellular compartment (ribosome, mitochondria)
o Cell (in many cases)
o Tissue
o Organism (example: yeast, not humans)
• Also the circumstances can differ
o Condition of the cell: age, differentiation, infection, tumor..
o Treatment: hypoxic conditions, virus infection,…
o Time after treatment: 15 min, 2 hours, 3 months, …
▪ Different time points need to be determined, because cell is dynamic and changes all the time
→ new proteins are released and the proteome changes
WHY PROTEOMICS?
1. Genomics vs proteomics
• Proteins are the workhorses of the cell
o Example: genomics are the different parts of a bicycle. When they work together you can ride it and investigate how it
works. This is wat proteomics does
• Proteogenomics
o Still difficult to predict some genes ➔ proteomics is needed to verify the gene product
2. mRNA vs protein profiling
• No direct correlation between amount of mRNA and protein abundancy!
• Microarrays are insufficient to measure protein expression
3. More proteins per gene: 1 gene can produce multiple proteins due to 2 reasons
• Post-translational modifications (PTM): addition/removal of specific groups
o Very dynamic processes
o Can’t see this via genomics
• Alternative splicing: different mRNA molecules out of 1 pre-mRNA result in multiple proteins
o Result: isoforms with (mostly) a high homology
o Can’t be predicted by genomics
o Example: α-1-antitrypsing has 22 different isoforms
4. Protein interaction networks
• = higher order of complexity without increase of number of components
• Most processes are regulated by protein complexes instead of individual proteins
o Not really the case for genes ➔ interaction can’t be predicted by genomics
o Important to know how the cell works, live,…
• Functional proteomics: protein as an element in an network (contextual function)
5. Cellular localization
• May be different depending of the biological state of the cell
o Nucleus, cytosol, ER, plasma,…
o Result: 1 protein can have several functions
• Example: interferon signalling pathway
o Jack and Stat interact with each other
o Depending on their place, this gives 2 different functions → Receptor recognition OR transcription factor
PROTEOMICS AS PART OF SYSTEMS BIOLOGY
To understand the dynamic complexity of an organism by looking at an integrated image of all aspects of proteins:
• mRNA & protein profiles → changing over time for example during pathology
• State & properties of proteins: PTMs, cellular localization, alternative splicing, proteolytic degradation,…
• Protein-protein interactions → in space and time in 1 cell
By combining this with genomic and metabolomic data, we can integrate this into system biology.
,THE DIFFERENT FACES OF PROTEOMICS
Proteomics can be divided into 3 groups:
• Proteomics sensu strictu: Large scale identification & characterization of proteins, inclusive their PTM
o Not very interesting, because just a description of the characteristics
o Need this when dealing with new organisms, but most proteomes are already described
• Differential proteomics: large scale comparison of protein expression levels
o Example: up-/downregulation of certain proteins when comparing healthy and cancer tissue
o May help to define biomarkers
• Cell-mapping proteomics: protein-protein interaction studies
IDENTIFICATION OF PROTEINS – THE PRINCIPLES
SAMPLE PREPARTION
Sample preparation is very important and you should always
be aware of what you are going to do next, because depending
on this the methods for separation will be different. In many
cases this is a trial and error process. The success of
separation/purification & identification is determined by
some important variables:
• Method of cell lysis, type of detergent
• pH → low: positively charged proteins ➔ negatively charge column needed
• Temperature → important for folding
• Proteolytic degradation → addition of protease inhibitors if you want to prevent this!
o Can be done before or after protein separation
o Hydrolysis of (specific) peptide bonds
▪ Trypsin: C-terminal of Lys/Arg
▪ Chymotrypsin: C-terminal of large hydrophobic residues (Trp, Phe, Tyr)
▪ Arg-C: cuts after Arg
▪ …
SEPARATION OF PROTEINS/PEPTIDES
Enrichment of proteins is needed, because there is a very wide dynamic range in a
cell, which makes is difficult to detect low abundant proteins. In order to increase
the separation capacity (peak capacity), sequential separation techniques are used
• Each dimension is based on different physicochemical characteristic
(= orthogonal separation)
o Example: 1st dimension is based on charge, 2nd on MW
• More separation techniques → increase of peak capacity
o Peak capacity = CS1 x CS2 x … (multiplication of peak capacities within a
certain chromatographic system)
2D-ELECTROPHORESIS (2D -PAGE)
2D-PAGE is a 2D separation technique based on the combination of isoelectric focusing (charge) & PAGE
(MW). As result a series of spots is obtained with each spot being one of a few proteins depending on the
complexity of the sample. Based on the x- and y-coordinates, the following characteristics can be
determined: pI, MW, PTM
CHROMATOGRAPHY
Chromatography is a separation technique based on the distribution of
biomolecules over a mobile and stationary phase
• Principle: molecules in mobile phase with lowest affinity for stationary
phase will be eluted first
• Different types depending of the stationary phase
• High Pressure Liquid Chromatography (HPLC)
o Solvent = mobile phase
o Column = stationary phase (beads)
o At a certain time point the sample is injected in which proteins
have different affinity
▪ Low affinity: eluted first
,ANALYSIS: MASS SPECTROMETRY
PROTEIN IDENTIFICATION BY DATA ANALYSIS
Based on 2 principles:
• De novo sequencing: direct determination of peptide AA sequence from the spectra
• In silico: comparing experiment MS spectra with in silico spectra generated from a protein database
o Requirement: gene must be present in the database
o PMF = peptide mass fingerprint
o PFF = peptide fragment fingerprint
WORKFLOW FOR ‘SHOTGUN’ PROTEOMICS
Bottom-up: immediately digesting the proteome into proteins
Top-down: immediately introducing the proteome mixture into MS
, LESSON 2 – SAMPLE PREPARATION (PROF. BOONEN)
Important principle: in = out ➔ bad sample gives bad results!
INTRODUCTION: PROTEOMICS IN LIFE SCIENCES (NOT IMPORTANT)
Proteins: molecules consisting of AA that perform much of life’s function
Proteome: set of all proteins in an organelle, cell, tissue, organ, organism or set of organisms
Proteomics: study of the proteome
• Studies needed to establish the identities, quantities, structures and biochemical & cellular functions of all proteins in an
organism, organ, organelle and how these properties vary in space, time of physiological state
• Multidisciplinary science
o Protein chemistry, mass spectrometry, genomics (proteogenomics), bioinformatics, computer & separation science
• Need it, because gene to a protein is not an one-to-one relationship
Proteoforms: different forms of the same protein arising from single nucleotide polymorphisms, alternative splicing of mRNA or
PTMs (phosphorylation, glycosylation,…)
• Techniques with high sensitivity & precision needed for the detection
• Important to understand diseases
When having complex samples, different things can be done.
• 2DGE & protein chips: intact proteins → proteoform info can be retained if spots are picked
o Gel-based separation
o Affinity purification by Ab
o Protein chips & arrays
o Ab-based identification or LC-MS/MS after separation
• MDLC: digested proteins → proteoform info is lost
o Liquid chromatography
o MS-based identification
Usually a separation step is done before MS, but it’s very difficult
to know which proteins and/or proteoforms were in a complex
sample once digested. This is called the protein interference
problem. Thus, the timing of digestion is important!
SAMPLE PREPARATION
There is diversity in the protein (domain) due to AA diversity: cytosolic & membrane associated proteins, protein complex, short
open reading frames. Because of this diversity, there are many different protocols and it becomes difficult to identify all proteins
• Extraction protocol is not exclusive
• Concentration range can be very different
• A more specific research question will result in more optimal preparation protocol
STEP 1: DEFINING YOUR RESEARCH QUESTION & LITERATURE STUDY
• What do you want to investigate? → proteins, proteoforms,…
• Which techniques are available and best suited?
• When will you do digestion?
• Needs the protein to be active?
• …
STEP 3: SAMPLE COLLECTION
Fresh samples are the best, because some proteins can degrade quickly. Different sample types can be used:
• Cell lines
o Possible to grown under controlled laboratory settings
o BUT not always representative of real tissues or cells (cellular dynamics)
• Patient samples
o More variable, because of differences in handling and in patients
o Large sample numbers needed to achieve statistical meaningful results
o Stabilization of the sample: usually freezing
• Serum and plasma
o Highly variable
o May be difficult to analyze, because of the large differences in protein concentration
, • Biobanked samples
o Can be highly variable
o Especially FFPE tissues
▪ FFPE = formalin-fixed paraffine embedded
▪ Hard to do proteomics on!
When taking a sample, a stressful situation is created and the proteome starts to degrade as response to this. Thus, the sample
need to be stabilized by inhibiting these degradation enzymes via:
• Phosphatase & protease inhibitors
• Cold temperature → cold can be reversible, heating usually not
• Adjustment of pH
• Chemical compounds (chaotropic agents & detergents) ➔ denaturation
STEP 4: PROTEIN EXTRACTION – SOLUBILIZATION
PROTEIN EXTRACTION
= bringing as much proteins as possible in solution via disruption of the cell membrane (and cell wall if present)
• Releasing all the proteins cannot be achieved
• Method depends on the sample type
o Plant, yeast, bacteria: really an effort to break the cell wall open
o Animal tissue
▪ Muscle tissue: you need to be sure that you get the proteins of the inside
▪ Brain tissue: here you have the issue that also a lot of fat etc. can be extracted
• Protein degradation must be prevented!
• Mechanical VS non-mechanical
o Mechanical: sonication, bead heating, mixing, freeze thawing
o Non-mechanical: detergents & chaotropes
• Soft VS harsh
o Soft: proteins will still be active
o Harsh: results in protein artefacts or denature of proteins
SOFT METHODS
• Osmotic shock
o Not the most effective way
o For cells that don’t have a rigid cell wall (example: bacteria)
• Most detergents
o Holes are made in the cell membrane ➔ proteins can get out
o Can also solubilize proteins in the membrane
• Enzymatic digestion
o Example: lysozyme → degradation of peptidoglycan (component of the cell wall of gram+ bacteria)
• Dounce homogenizer = glass tube with a small clearance between the head and tube
o Cooled solution containing the cells (minimal heating!) is forced in between the tube and the homogenizer which
causes a friction to break the cells (opening by shearing)
o Possible to play with the clearance
o Possible to keep the organelles intact, such as the nucleus
o Only used for tissues & cells that can be easily extracted
HARSH METHODS
• Blender, tissue chopper (for tissues)
• Cryo-grinding: sample is frozen by liquid N2 for example and then pulverized with a pestle and mortar
o ! Minimal heating
• Bead heater: controlled way to pulverize sample → you can play with the intervals
• Sonication: very harsh method
o ! Sample need to be kept on ice in between pulses
As a result, you have a sample with proteins in solution and remnants of the cells. It’s also possible to obtain the subcellular
fraction to get more info about: plasma membrane, nucleus, ribosomes, exosomes, mitochrondria, Golgi apparatus, lysozomes
,PROTEIN SOLUBILIZATION
Protein solubilization is a part of the protein extraction.
• Aim: maximal solubility of all proteins
• Choice of cell lysis buffer for extraction is crucial!
• Denaturing VS non-denaturing
o Denaturing: non-covalent & disulfide bonds are broken ➔ no biological activity
o Non-denaturing ➔ biological activity of proteins is retained
CHAOTROPIC AGENTS
• Increase of entropy & decrease of net hydrophobic effect ➔ denaturation
• Chaotropic agents will
o Change hydrogen-bonding in the solvent
o Disrupt hydrogen-bonding in proteins
o Lower energy barrier for exposing apolar groups
• Examples:
o Urea (7-8 M)→ good for 2D-PAGE, less good for hydrophobic disruption
o Thio-urea (2 M) → can penetrate into the membrane
o Guanidinium chloride (6 M) → H-bond disruption & hydrophobic region disruption
DETERGENTS (OR SURFACTANTS)
Detergents are amphipathic, meaning they have a hydrophobic and hydrophilic region, and will form micelles with a hydrophobic
core, which associates with the hydrophobic region of the proteins.
• Detergent concentration should be at least 2x the critical micelle concentration (CMC)
• Can be denaturing or non-denaturing
o Anionic detergents (denaturing)
▪ Contain an anionic headgroup (sulphate, sulphonate, phosphate carboxylate)
▪ Disruption of membranes, protein-protein interactions & protein activity
▪ Example: SDS = sodium dodecyl sulphate
- Cooperative binding!
- If a protein binds to SDS, the probability increases that another
molecule of SDS will bind to the same protein
o Non-ionic detergents (non-denaturing)
▪ Disruption of lipid-lipid & lipid-protein interactions (NOT protein-protein interactions)
▪ Application: isolation of membrane proteins in their biologically active form
▪ Examples: Tween, Triton, Maltoside, NP40, Octyl-thioglucoside
o Zwitterionic detergents (non-denaturing)
▪ Netto charge = 0, but polar
▪ Disruption of protein-protein interactions
▪ Application: isoelectric focusing & ion-exchange chromatography
▪ Example: CHAPS
• Need to be removed before digestion, because it will interfere with MS analysis → different options
o Acetone (protein) precipitation → denaturing
o Mass cut-off filter → easy & quick (1 centrifugation step)
o Gel filtration chromatography → buffer contaminants are retained on the gel
o Dialysis
STEP 5: PROTEIN SEPARATION OR PURIFICATION (OPTIONAL)
STEP 6: REDUCTION AND ALKYLATION
Disulfide bridges need to be broken prior to digestion and LC-MS analysis
• β-mercaptoethanol
o > 700 mM ➔ affecting IEF-basic gradient region
o Ionization at high pH
• DTT = dithiothreitol
o 50 mM
o Ionization at high pH , BUT not affection IEF gradients due to lower concentration
When opening up the disulfide bonds, you create highly reactive thiol-groups. These need to be stabilized by adding a group on
the Cys-residues so it can’t reform (= thiol group chemical stabilization). This is mostly done by iodoacetic acid or iodoacetamide.
• Iodoacetic acid → carboxymethylation ➔ increase of mass by 58.005 Da
• Iodoacetamide → carbamidomethylation ➔ increase of mass by 57.021 Da
• Practical course: maleimide ➔ increase of mass by 97.016 Da
, In order to deal with the protein abundancy, two things can be done (optional)
• High abundant protein depletion
o Why: LC-MS has limited concentration detection ➔ low abundant proteins will not be detected
o Example: depletion of high abundant proteins with Ab (immunodepletion)
• Low abundant protein depletion
o Example: ProteoMiner beads
o Beads coated with peptide hexamers, which bind to highly abundant proteins ➔ saturation & wash away of non-bound
proteins ➔ smaller concentration differences
STEP 7: DIGESTION
Usually, trypsin is used, because:
• Cleaves proteins into peptide of 10 – 20 AA length (ideal of LC-MS)
• Cleaves after Lys or Arg (good for ionization)
• Digestion: 1:100 trypsin/protein ratio → usually overnight at 37 °C
o Can be combined with LysC for more complete digestion
• Active up to 2M urea
• BUT only suitable when enough trypsin cleavage sites are available
After digestion, you can do peptide labeling for quantitative proteomics (optional).
Usually amine reactive groups are used.
STEP 8: PEPTIDES PURIFICATION OR SELECTION
Detergents, salts, protease inhibitors and other compounds need to be removed before LC-MS analysis. On protein level, this can
be done by precipitation, gelfiltration, SDS-PAGE, dialysis or filtering. On peptide level, this is done by C18 solid phase extraction
or ion-exchange chromatography to remove digestion or peptide labelling buffer.
C18 SOLID PHASE EXTRACTION ( R E V E R S E D P H A S E L I Q U I D C H R O M A T O G R A P H Y )
• Separation according to hydrophobicity
• Covalent attachment of stationary phase ➔ thermally/hydrolytically stable bonded phase
• Larger polypeptides: C8 or C4
• Elution by fixed acetonitrile concentration (gradient can also be used)
• Less polar → eluted first
• Afterward: drying to remove acetonitrile!
STRONG CATION EXCHANGE (SCX) CHROMATOGRAPHY
• Why: adding labels increases the charge on a peptide to enhance ionization
• Positively charged peptides will bind to negatively charged beads
• Elution with ammonium nitrate (needs also to be removed afterwards!)
STEP 9: PREFRACTIONING (OPTIONAL)
Can be done to simplify the peptide sample before you go to LC-MC. Important is that it needs to be orthogonal to the last LC
step! Thus, for MS you use a low pH, for RP-LC a high pH can be used.
