2025-2026 Concepts of Protein Technology Xaveer Van Ostade
CONCEPTS OF PROTEIN
TECHNOLOGY & PROTEOME ANALYSIS
CHAPTER 1: INTRODUCTION
WHAT IS PROTEOMICS?
• Proteomics = determination of the complete set of proteins that is present in a system under specific
circumstances.
1. System = protein complex / cell / tissue / organism
2. Specific circumstances = a treatment / time after treatment / condition cell like hypoxia or
apoptosis
• Proteome = set of all proteins in an organelle, cell, tissue, organ, organism
1. Meta-proteome = set of all proteins in a set of organisms e.g. the gut
• Why would we use proteomics for studies (we already have genomics?)
1. Difficult to predict the genes, verification of gene product by proteomic analysis is still
necessary = proteogenomics
§ Genes need to be converted in something functional = difficult to predict how the
genes will develop
§ Humans have around 20.000-40.000 genes while earth worms have 18.000 2. mRNAgenes àprofiling
vs. Protein
while we don’t have that much more genes, we are more complex = genes do not
No direct correlation: microarrays/RNAseq are insufficient to measure protein
expression.
give information about the complexity of an organism
2. Sometimes mRNA is high but protein expression low and the other way around
= protein profiling
3. Sometimes one gene can code for multiple proteins due to PTM or alternative
splicing (forming isoforms) or nucleotide polymorphisms
§ This can lead to different proteoforms/isoforms of protein: several forms/versions
of proteins coming from the same gene (malfunctioning of isoform cannot be seen in
DNA but only with proteomics)
4. A protein never works alone, but in a network. The cellular processes are regulated by these
networks = functional proteomics (=proteins as an element in an interaction network
instead of ascribing it to one function)
§ You can use ‘guilt by association’ meaning that if you don’t know the function of 1
protein, you can assume its function based on the network it works in
5. With proteomics we can see the cellular localization of a protein
§ Depending on the biological state of the cell, a protein can be localized in 1 or
different cellular locations
§ Different bin ding partners in different locations; a protein might have a different
function depending on its localization
• Proteomics are a part in the study of systems biology: we want to understand the dynamic
complexity of an organism à for this we need to understand the proteins which control almost every
process in life. Proteins are dynamic = they constantly change (folding, binding, unfolding,
interacting…) à an integrated image of all aspects of proteins needs to be developed (so far, we can
1
,2025-2026 Concepts of Protein Technology Xaveer Van Ostade
only measure the average of all possible states = we can see a hazy blur that reflects the average of
all positions, but the precise steps, transitions… are lost = we miss the fine-grained dynamics that
truly define protein behavior).
o mRNA and protein profiles and how these change over time
o Knowledge of the state and properties of all proteins (PTM, cellular localization, AS,
structure and conformations…)
o All protein-protein interactions in space and time in 1 cell
o Together with genomic and metabolomic data = systems biology
• Different faces of proteomics:
o Proteomics sensu strictu: large identification and characterization of proteins inclusive of
their PTM
§ Shotgun proteomics = identification and characterization of as many proteins as
possible
o Differential proteomics: comparison of proteins (expression levels) on a large scale
o Cell mapping proteomics: protein-protein interactions are studied
IDENTIFICATION OF PROTEINS: PRINCIPLES
1. Protein extraction from sample
2. If you wish you can already separate proteins (SDS-page or LC)
3. Digest proteins with proteases to obtain peptides
4. Separate the peptides using liquid chromatography (LC) (Not SDS since this can only separate
proteins)
5. Ionize the peptides using electrospray ionization
6. Not mandatory: we can measure ion mobility by separating the ions before they reach the MS
7. Mass spectrometry (mostly tandem MS)
8. Analysis: Bioinformatics!
V. Identification of proteins:
principles
If you do SDS, can you still use the sample afterward? Yes. We can cut out a band from the gel, digest it with
enzymes (e.g., trypsin), and analyze the peptides using mass spectrometry.
Bioinformatics
Separation of Functional
Sample preparation analysis Identification
proteins/peptides Analysis
Preparation of GO/Pathway
protein fraction PAGE/HPLC Mass spectrometry Database search
analysis
Applied bio-
informatics
2
,2025-2026 Concepts of Protein Technology Xaveer Van Ostade
SAMPLE PREPARATION
• Very important step: you can have the best machine, but if the sample you bring in is crap, the results
will be crap.
1. First the cells/ tissue must be lysed for the proteins to come free (see later), then the proteins
must be extracted from sample that now also contains other cell debris
2. You can optionally separate proteins (SDS-page or LC)
à This is needed if you want to study different proteoforms: once you have digested the
proteoforms you don’t know what peptides belong to what proteoforms. If you want to
differentiate between them, you will have to separate them before tryptic digest.
3. If this is needed for further analysis: modify your protein
à Denature or reduce your protein
à Depending on the forthcoming methods for separation/purification and identification (e.g.
when you need the native protein, you can’t use trypsin (protease))
4. The success of separation/purification and identification is determined by:
a. Method of cell lysis, type of detergent
b. pH
c. temperature
d. proteolytic degradation (addition of protease inhibitors)
à Proteases are normally localized in certain compartments of cell where they cut
proteins. However, once the cell is lysed the proteases are free to cut everywhere. To
stop this: protease inhibitors
5. Often: trial and error
6. To prepare the proteins for MS analysis we need to do a digestion = protease treatment (can be
done before or after the protein separation)
a. Trypsin cuts at C-terminal site of the amino acids: Lysine and Arginine. These are
positively charged and give a good length of peptides (10-20AA)
b. There are other proteases like chymotrypsin (cuts large hydrophobic aa like Tyr, Phe, Trp)
c. Depending on the protease you use, you will have different sizes of peptides
SEPARATION OF PROTEINS OR PEPTIDES
• Whether you do a separation of proteins or peptides depends on if you separate before or after
digestion
• Why do we do protein/ peptide separation?
o We need enrichment of the proteins/ peptides. The dynamic range of the abundance of a
protein in a cell is very wide: 10 to 100.000 copies per cell. This makes the detection of low
abundant proteins very difficult: for MS the proteins will be ionized, and the high abundant
proteins will suppress the ionization and detection of the low abundant proteins.
o The figure shows this:
§ Coomassie detects proteins in the nM range.
It only detects very abundant proteins (only
the dark blue line with 1000-100.000 protein
copies/cell)
§ Silverstaining detects in the high fM range, but
low abundant proteins (10 copies/cell) are
only detected if the number of cells that are
loaded on the gel is very high (1x1010 cells!!!)
3
, 2025-2026 Concepts of Protein Technology Xaveer Van Ostade
o These days many MS will measure in the aM and fM range, but if the proteins are not
abundant enough, they will still not be measured.
• In plasma, the concentrations of different proteins differ by 11 orders of magnitude = extreme
difference. When protein abundancy is very low (like the abundancy of interleukins in plasma) we can
combine different sequential separation techniques = “dimensions” ( e.g. SDS + LC performed
sequentially). Each dimension is based on a different physicochemical characteristic of the proteins
or peptides (orthogonal separation).
o The separation capacity = multiply the peak capacity of each technique
o For e.g. SDS has peak capacity of 5 and LC has peak capacity of 6. The total separation
capacity: 5 x 6 = 30!
Peak capacity = the maximum number of peaks that can be separated (without overlap) and resolved in a given
separation method (e.g. chromatography or SDS)
CHROMATOGRAPHY
• Usually used for peptide separation
• Chromatography = separation of biomolecules based on their distribution over a mobile and a
stationary phase
• High Pressure Liquid Chromatography (HPLC)
o Pump: pushes the solvent (mobile
phase) through the system at high pressure
o Injector: introduces the sample into the flowing
solvent stream
o Column: packed with beads = stationary phase
à separates the mixture’s components based on
their chemical interactions and retention times
o Detector (MS): instead of a regular UV detector,
this setup uses a Mass Spectrometer (MS) to
detect and identify the separated compounds
based on their mass-to-charge ratio (m/z)
o Data system: collects and displays chromatograms and mass spectra for analysis
o Waste: excess solvent and non-analyzed material are discarded here
• We can also skip the detector and go directly to MS = online LC-MS
• Different Types of chromatograms:
o Ion -exchange:
§ Stationary phase is positively or negatively charged
§ If it is positively charged = anion exchange
§ If it is negatively charged = cation exchange
§ The elution buffer contains salt to wash the proteins that bind to stationary phase away
§ Selection of proteins based on charge
o Gel permeation/ gel filtration
§ Stationary phase is beads, beads have a certain pore-diameter
§ The mobile phase are proteins in solvent, the proteins that are small enough to pass
through pores are eluted slower while bigger proteins will not pass through the pores
and be eluted quicker (small proteins travel a longer distance)
§ Selection of proteins based on their mass
4
CONCEPTS OF PROTEIN
TECHNOLOGY & PROTEOME ANALYSIS
CHAPTER 1: INTRODUCTION
WHAT IS PROTEOMICS?
• Proteomics = determination of the complete set of proteins that is present in a system under specific
circumstances.
1. System = protein complex / cell / tissue / organism
2. Specific circumstances = a treatment / time after treatment / condition cell like hypoxia or
apoptosis
• Proteome = set of all proteins in an organelle, cell, tissue, organ, organism
1. Meta-proteome = set of all proteins in a set of organisms e.g. the gut
• Why would we use proteomics for studies (we already have genomics?)
1. Difficult to predict the genes, verification of gene product by proteomic analysis is still
necessary = proteogenomics
§ Genes need to be converted in something functional = difficult to predict how the
genes will develop
§ Humans have around 20.000-40.000 genes while earth worms have 18.000 2. mRNAgenes àprofiling
vs. Protein
while we don’t have that much more genes, we are more complex = genes do not
No direct correlation: microarrays/RNAseq are insufficient to measure protein
expression.
give information about the complexity of an organism
2. Sometimes mRNA is high but protein expression low and the other way around
= protein profiling
3. Sometimes one gene can code for multiple proteins due to PTM or alternative
splicing (forming isoforms) or nucleotide polymorphisms
§ This can lead to different proteoforms/isoforms of protein: several forms/versions
of proteins coming from the same gene (malfunctioning of isoform cannot be seen in
DNA but only with proteomics)
4. A protein never works alone, but in a network. The cellular processes are regulated by these
networks = functional proteomics (=proteins as an element in an interaction network
instead of ascribing it to one function)
§ You can use ‘guilt by association’ meaning that if you don’t know the function of 1
protein, you can assume its function based on the network it works in
5. With proteomics we can see the cellular localization of a protein
§ Depending on the biological state of the cell, a protein can be localized in 1 or
different cellular locations
§ Different bin ding partners in different locations; a protein might have a different
function depending on its localization
• Proteomics are a part in the study of systems biology: we want to understand the dynamic
complexity of an organism à for this we need to understand the proteins which control almost every
process in life. Proteins are dynamic = they constantly change (folding, binding, unfolding,
interacting…) à an integrated image of all aspects of proteins needs to be developed (so far, we can
1
,2025-2026 Concepts of Protein Technology Xaveer Van Ostade
only measure the average of all possible states = we can see a hazy blur that reflects the average of
all positions, but the precise steps, transitions… are lost = we miss the fine-grained dynamics that
truly define protein behavior).
o mRNA and protein profiles and how these change over time
o Knowledge of the state and properties of all proteins (PTM, cellular localization, AS,
structure and conformations…)
o All protein-protein interactions in space and time in 1 cell
o Together with genomic and metabolomic data = systems biology
• Different faces of proteomics:
o Proteomics sensu strictu: large identification and characterization of proteins inclusive of
their PTM
§ Shotgun proteomics = identification and characterization of as many proteins as
possible
o Differential proteomics: comparison of proteins (expression levels) on a large scale
o Cell mapping proteomics: protein-protein interactions are studied
IDENTIFICATION OF PROTEINS: PRINCIPLES
1. Protein extraction from sample
2. If you wish you can already separate proteins (SDS-page or LC)
3. Digest proteins with proteases to obtain peptides
4. Separate the peptides using liquid chromatography (LC) (Not SDS since this can only separate
proteins)
5. Ionize the peptides using electrospray ionization
6. Not mandatory: we can measure ion mobility by separating the ions before they reach the MS
7. Mass spectrometry (mostly tandem MS)
8. Analysis: Bioinformatics!
V. Identification of proteins:
principles
If you do SDS, can you still use the sample afterward? Yes. We can cut out a band from the gel, digest it with
enzymes (e.g., trypsin), and analyze the peptides using mass spectrometry.
Bioinformatics
Separation of Functional
Sample preparation analysis Identification
proteins/peptides Analysis
Preparation of GO/Pathway
protein fraction PAGE/HPLC Mass spectrometry Database search
analysis
Applied bio-
informatics
2
,2025-2026 Concepts of Protein Technology Xaveer Van Ostade
SAMPLE PREPARATION
• Very important step: you can have the best machine, but if the sample you bring in is crap, the results
will be crap.
1. First the cells/ tissue must be lysed for the proteins to come free (see later), then the proteins
must be extracted from sample that now also contains other cell debris
2. You can optionally separate proteins (SDS-page or LC)
à This is needed if you want to study different proteoforms: once you have digested the
proteoforms you don’t know what peptides belong to what proteoforms. If you want to
differentiate between them, you will have to separate them before tryptic digest.
3. If this is needed for further analysis: modify your protein
à Denature or reduce your protein
à Depending on the forthcoming methods for separation/purification and identification (e.g.
when you need the native protein, you can’t use trypsin (protease))
4. The success of separation/purification and identification is determined by:
a. Method of cell lysis, type of detergent
b. pH
c. temperature
d. proteolytic degradation (addition of protease inhibitors)
à Proteases are normally localized in certain compartments of cell where they cut
proteins. However, once the cell is lysed the proteases are free to cut everywhere. To
stop this: protease inhibitors
5. Often: trial and error
6. To prepare the proteins for MS analysis we need to do a digestion = protease treatment (can be
done before or after the protein separation)
a. Trypsin cuts at C-terminal site of the amino acids: Lysine and Arginine. These are
positively charged and give a good length of peptides (10-20AA)
b. There are other proteases like chymotrypsin (cuts large hydrophobic aa like Tyr, Phe, Trp)
c. Depending on the protease you use, you will have different sizes of peptides
SEPARATION OF PROTEINS OR PEPTIDES
• Whether you do a separation of proteins or peptides depends on if you separate before or after
digestion
• Why do we do protein/ peptide separation?
o We need enrichment of the proteins/ peptides. The dynamic range of the abundance of a
protein in a cell is very wide: 10 to 100.000 copies per cell. This makes the detection of low
abundant proteins very difficult: for MS the proteins will be ionized, and the high abundant
proteins will suppress the ionization and detection of the low abundant proteins.
o The figure shows this:
§ Coomassie detects proteins in the nM range.
It only detects very abundant proteins (only
the dark blue line with 1000-100.000 protein
copies/cell)
§ Silverstaining detects in the high fM range, but
low abundant proteins (10 copies/cell) are
only detected if the number of cells that are
loaded on the gel is very high (1x1010 cells!!!)
3
, 2025-2026 Concepts of Protein Technology Xaveer Van Ostade
o These days many MS will measure in the aM and fM range, but if the proteins are not
abundant enough, they will still not be measured.
• In plasma, the concentrations of different proteins differ by 11 orders of magnitude = extreme
difference. When protein abundancy is very low (like the abundancy of interleukins in plasma) we can
combine different sequential separation techniques = “dimensions” ( e.g. SDS + LC performed
sequentially). Each dimension is based on a different physicochemical characteristic of the proteins
or peptides (orthogonal separation).
o The separation capacity = multiply the peak capacity of each technique
o For e.g. SDS has peak capacity of 5 and LC has peak capacity of 6. The total separation
capacity: 5 x 6 = 30!
Peak capacity = the maximum number of peaks that can be separated (without overlap) and resolved in a given
separation method (e.g. chromatography or SDS)
CHROMATOGRAPHY
• Usually used for peptide separation
• Chromatography = separation of biomolecules based on their distribution over a mobile and a
stationary phase
• High Pressure Liquid Chromatography (HPLC)
o Pump: pushes the solvent (mobile
phase) through the system at high pressure
o Injector: introduces the sample into the flowing
solvent stream
o Column: packed with beads = stationary phase
à separates the mixture’s components based on
their chemical interactions and retention times
o Detector (MS): instead of a regular UV detector,
this setup uses a Mass Spectrometer (MS) to
detect and identify the separated compounds
based on their mass-to-charge ratio (m/z)
o Data system: collects and displays chromatograms and mass spectra for analysis
o Waste: excess solvent and non-analyzed material are discarded here
• We can also skip the detector and go directly to MS = online LC-MS
• Different Types of chromatograms:
o Ion -exchange:
§ Stationary phase is positively or negatively charged
§ If it is positively charged = anion exchange
§ If it is negatively charged = cation exchange
§ The elution buffer contains salt to wash the proteins that bind to stationary phase away
§ Selection of proteins based on charge
o Gel permeation/ gel filtration
§ Stationary phase is beads, beads have a certain pore-diameter
§ The mobile phase are proteins in solvent, the proteins that are small enough to pass
through pores are eluted slower while bigger proteins will not pass through the pores
and be eluted quicker (small proteins travel a longer distance)
§ Selection of proteins based on their mass
4
