ADVANCED PROTEIN TECHNOLOGY AND
PROTEOME ANALYSIS
INHOUDSOPGAVE
1.1 Proteomics ...................................................................................................................................... 1
1.1.1 Bottom-up approach............................................................................................................................. 1
1.1.2 Shotgun proteomics .............................................................................................................................. 1
1.1.3 Sample preparation .............................................................................................................................. 2
1.1.4 Trends in proteomics ............................................................................................................................ 3
1.2 Sample prep protocols (weten waarom, probleem oplossen met deze techniek en waarom?) ............ 4
1.2.1 Filter aided-sample preparation (FASP) ................................................................................................ 4
1.2.2 Paramagnetic beads ............................................................................................................................. 5
1.2.3 Minimal, encapsulated proteomic-sample processing ......................................................................... 5
1.2.4 Suspension trapping ............................................................................................................................. 6
1.2.5 Nano-POTS (nanodroplet processing in one pot for trace samples) .................................................... 7
1.2.6 On-Chip Sample Preparation Using a ChipFilter Coupled to NanoLCMS/MS for Bottom-Up
Proteomics ............................................................................................................................................................ 7
1.2.7 iPAD and iPAD-1: integrated proteome analysis device ....................................................................... 7
1.2.8 Lab-on-a-chip ........................................................................................................................................ 8
1.3 Reproducibility ................................................................................................................................ 8
1.4 Proteomic applications (begrijpen, geen details van protocollen) ...................................................... 9
1.4.1 Shotgun proteomics .............................................................................................................................. 9
1.4.2 COFRADIC ............................................................................................................................................ 10
1.4.3 Biotin-avidin reactions ........................................................................................................................ 11
1.4.4 Interactomics ...................................................................................................................................... 12
1.4.5 Post translational modifications ......................................................................................................... 12
1.4.6 Complex proteomics ........................................................................................................................... 13
1.4.7 Immunopeptidomics ........................................................................................................................... 14
2.1 Adapted mass spectrometers for the study of protein complexes .................................................... 15
2.1.1 Mass spectrometers for measurement of large proteins ................................................................... 15
2.2 Ion-mobility mass spectrometry (IMS) ............................................................................................ 17
1
, 2.2.1 Principle .............................................................................................................................................. 17
2.2.2 Different types of IMS ......................................................................................................................... 17
2.2.2.1 Drift time IMS (DTIMS) or Aspiration IMS (AIMS) ...................................................................... 17
2.2.2.2 Alternating field IMS (AFIMS) or Field-asymmetric waveform IMS (FAIMS) ............................. 18
2.2.2.3 Traveling wave IMS (TWIMS) ...................................................................................................... 18
2.2.2.4 Summary ..................................................................................................................................... 19
2.2.3 An additional level of separation ........................................................................................................ 19
3.1 Cystine bridge formation ............................................................................................................... 21
3.2 Phosphoproteomics ....................................................................................................................... 21
3.2.1 General................................................................................................................................................ 21
3.2.2 Enrichment of phosphopeptides ......................................................................................................... 22
3.2.2.1 Immuno affinity chromatography .............................................................................................. 22
3.2.2.2 Immobilised metal affinity chromatography (IMAC) ................................................................. 22
3.2.2.3 Titanium dioxide chromatography ............................................................................................. 23
3.2.2.4 SCX chromatography .................................................................................................................. 23
3.2.2.5 Chemical derivatization .............................................................................................................. 23
3.2.2.6 Overview of enrichment strategies for phosphopeptides ......................................................... 24
3.2.3 Phosphopeptide determination by MS/MS ........................................................................................ 24
3.3 Glycoproteomics ........................................................................................................................... 26
3.3.1 General................................................................................................................................................ 26
3.3.2 Enrichment of glycoproteins by lectin affinity chromatography ........................................................ 27
3.3.3 Separation of glycoproteins by hydrophilic Interaction Chromatography (HILIC).............................. 27
3.3.4 Localisation of glycosylation by mass spectrometry .......................................................................... 28
4.1 What is the metabolome?.............................................................................................................. 30
4.2 Metabolomics ............................................................................................................................... 30
4.2.1 Introduction ........................................................................................................................................ 30
4.2.2 Metabolomic complexity .................................................................................................................... 31
4.2.3 Metabolomic dynamics....................................................................................................................... 31
4.2.4 Metabolomics – future relevance ....................................................................................................... 32
4.2.5 Steps at metabolomics........................................................................................................................ 32
4.3 Nuclear magnetic resonance (NMR) ............................................................................................... 33
4.3.1 Introduction (background knowledge) ............................................................................................... 33
4.3.2 MS versus NMR ................................................................................................................................... 34
4.4 LC-MS based metabolomics vs. proteomics..................................................................................... 34
4.5 Generic Metabolomics studies ....................................................................................................... 35
2
, 4.5.1 The targeting dichotomy .................................................................................................................... 35
4.5.2 Targeted metabolomics ...................................................................................................................... 36
4.5.3 The metabolomics pipeline ................................................................................................................. 37
4.6 Experimental design ...................................................................................................................... 37
4.6.1 Considerations .................................................................................................................................... 37
4.6.2 Sample preparation ............................................................................................................................ 37
4.6.3 Extractions – chemical and physical properties.................................................................................. 38
4.6.4 Quenching – Stop enzymatic metabolism .......................................................................................... 38
4.6.5 Mass spectrometry (MS) ..................................................................................................................... 39
4.6.6 Separation – HPLC .............................................................................................................................. 40
4.6.7 Data processing .................................................................................................................................. 41
4.6.8 Analysis and interpretation ................................................................................................................ 44
4.7 Take home points .......................................................................................................................... 44
5.1 Definitions .................................................................................................................................... 45
5.2 Molecules involved........................................................................................................................ 45
5.3 Size ............................................................................................................................................... 46
5.4 Defining PPIs ................................................................................................................................. 46
5.4.1 In general ............................................................................................................................................ 46
5.4.2 Large scale protein-protein interaction determination ...................................................................... 47
5.4.2.1 Yeast-Two-Hybrid and variants .................................................................................................. 47
5.4.2.2 Tandem affinity procedure (TAP) ............................................................................................... 48
5.4.2.3 Proximity labeling ....................................................................................................................... 49
5.5 Biophysical methods to confirm PPIs .............................................................................................. 52
5.5.1 Surface Plasmon resonance (SPR) ...................................................................................................... 52
5.5.2 Bio-Layer Interferometry (BLI) ............................................................................................................ 53
5.6 Protein-protein interactomes: Databases and Applications ............................................................. 54
5.6.1 Databases ........................................................................................................................................... 54
5.6.2 Validation of interactomes ................................................................................................................. 55
5.6.3 Analysis of interactome networks ...................................................................................................... 55
5.6.4 Some interactomes (no details) .......................................................................................................... 56
5.6.5 Some applications ............................................................................................................................... 57
5.6.6 The SARS-CoV-2 interactome (AP-MS studies) ................................................................................... 57
3
, CH 1: SAMPLE PREPARATION (KURT BOONEN)
1.1 PROTEOMICS
1.1.1 BOTTOM-UP APPROACH
The most commonly used proteomics technique is
bottom-up.
Proteins are digested into smaller peptides to identify
the peptides based on their fragmentation spectrum.
Proteins can be measured intact, but this is
significantly more difficult since the interpretation of
protein fragmentation spectra is hard. It is much
easier to interpret data from peptide analysis than
from complete proteins top-down approach, where
proteins are left intact: electrospray ionization (ESI) –
intact precursor ion – fragmentation.
A complex protein sample, results after digestion in a
complex peptide mix, is often separated by
electrophoresis or chromatography. Then, MS or
MS/MS analysis can be done on a simpler sample
(protein/peptide). Which is selected? Most abundant?
Think about this when conducting an experiment.
1.1.2 SHOTGUN PROTEOMICS
Shotgun proteomics acquire as much fragmentation spectra as possible from a sample and identify them! If the
MS software selects the peptides for fragmentation based on what is detected in MS1 we call it data dependent
acquisition (DDA). Shotgun is usually bottom-up.
After digestion, we have a
mixture – separated with LC –
then with MS1 see what is in it –
fragmentation.
Data-dependent acquisition
(DDA) = when the MS software
selects peptides for further
fragmentation based on what is
detected in MS1.
1
PROTEOME ANALYSIS
INHOUDSOPGAVE
1.1 Proteomics ...................................................................................................................................... 1
1.1.1 Bottom-up approach............................................................................................................................. 1
1.1.2 Shotgun proteomics .............................................................................................................................. 1
1.1.3 Sample preparation .............................................................................................................................. 2
1.1.4 Trends in proteomics ............................................................................................................................ 3
1.2 Sample prep protocols (weten waarom, probleem oplossen met deze techniek en waarom?) ............ 4
1.2.1 Filter aided-sample preparation (FASP) ................................................................................................ 4
1.2.2 Paramagnetic beads ............................................................................................................................. 5
1.2.3 Minimal, encapsulated proteomic-sample processing ......................................................................... 5
1.2.4 Suspension trapping ............................................................................................................................. 6
1.2.5 Nano-POTS (nanodroplet processing in one pot for trace samples) .................................................... 7
1.2.6 On-Chip Sample Preparation Using a ChipFilter Coupled to NanoLCMS/MS for Bottom-Up
Proteomics ............................................................................................................................................................ 7
1.2.7 iPAD and iPAD-1: integrated proteome analysis device ....................................................................... 7
1.2.8 Lab-on-a-chip ........................................................................................................................................ 8
1.3 Reproducibility ................................................................................................................................ 8
1.4 Proteomic applications (begrijpen, geen details van protocollen) ...................................................... 9
1.4.1 Shotgun proteomics .............................................................................................................................. 9
1.4.2 COFRADIC ............................................................................................................................................ 10
1.4.3 Biotin-avidin reactions ........................................................................................................................ 11
1.4.4 Interactomics ...................................................................................................................................... 12
1.4.5 Post translational modifications ......................................................................................................... 12
1.4.6 Complex proteomics ........................................................................................................................... 13
1.4.7 Immunopeptidomics ........................................................................................................................... 14
2.1 Adapted mass spectrometers for the study of protein complexes .................................................... 15
2.1.1 Mass spectrometers for measurement of large proteins ................................................................... 15
2.2 Ion-mobility mass spectrometry (IMS) ............................................................................................ 17
1
, 2.2.1 Principle .............................................................................................................................................. 17
2.2.2 Different types of IMS ......................................................................................................................... 17
2.2.2.1 Drift time IMS (DTIMS) or Aspiration IMS (AIMS) ...................................................................... 17
2.2.2.2 Alternating field IMS (AFIMS) or Field-asymmetric waveform IMS (FAIMS) ............................. 18
2.2.2.3 Traveling wave IMS (TWIMS) ...................................................................................................... 18
2.2.2.4 Summary ..................................................................................................................................... 19
2.2.3 An additional level of separation ........................................................................................................ 19
3.1 Cystine bridge formation ............................................................................................................... 21
3.2 Phosphoproteomics ....................................................................................................................... 21
3.2.1 General................................................................................................................................................ 21
3.2.2 Enrichment of phosphopeptides ......................................................................................................... 22
3.2.2.1 Immuno affinity chromatography .............................................................................................. 22
3.2.2.2 Immobilised metal affinity chromatography (IMAC) ................................................................. 22
3.2.2.3 Titanium dioxide chromatography ............................................................................................. 23
3.2.2.4 SCX chromatography .................................................................................................................. 23
3.2.2.5 Chemical derivatization .............................................................................................................. 23
3.2.2.6 Overview of enrichment strategies for phosphopeptides ......................................................... 24
3.2.3 Phosphopeptide determination by MS/MS ........................................................................................ 24
3.3 Glycoproteomics ........................................................................................................................... 26
3.3.1 General................................................................................................................................................ 26
3.3.2 Enrichment of glycoproteins by lectin affinity chromatography ........................................................ 27
3.3.3 Separation of glycoproteins by hydrophilic Interaction Chromatography (HILIC).............................. 27
3.3.4 Localisation of glycosylation by mass spectrometry .......................................................................... 28
4.1 What is the metabolome?.............................................................................................................. 30
4.2 Metabolomics ............................................................................................................................... 30
4.2.1 Introduction ........................................................................................................................................ 30
4.2.2 Metabolomic complexity .................................................................................................................... 31
4.2.3 Metabolomic dynamics....................................................................................................................... 31
4.2.4 Metabolomics – future relevance ....................................................................................................... 32
4.2.5 Steps at metabolomics........................................................................................................................ 32
4.3 Nuclear magnetic resonance (NMR) ............................................................................................... 33
4.3.1 Introduction (background knowledge) ............................................................................................... 33
4.3.2 MS versus NMR ................................................................................................................................... 34
4.4 LC-MS based metabolomics vs. proteomics..................................................................................... 34
4.5 Generic Metabolomics studies ....................................................................................................... 35
2
, 4.5.1 The targeting dichotomy .................................................................................................................... 35
4.5.2 Targeted metabolomics ...................................................................................................................... 36
4.5.3 The metabolomics pipeline ................................................................................................................. 37
4.6 Experimental design ...................................................................................................................... 37
4.6.1 Considerations .................................................................................................................................... 37
4.6.2 Sample preparation ............................................................................................................................ 37
4.6.3 Extractions – chemical and physical properties.................................................................................. 38
4.6.4 Quenching – Stop enzymatic metabolism .......................................................................................... 38
4.6.5 Mass spectrometry (MS) ..................................................................................................................... 39
4.6.6 Separation – HPLC .............................................................................................................................. 40
4.6.7 Data processing .................................................................................................................................. 41
4.6.8 Analysis and interpretation ................................................................................................................ 44
4.7 Take home points .......................................................................................................................... 44
5.1 Definitions .................................................................................................................................... 45
5.2 Molecules involved........................................................................................................................ 45
5.3 Size ............................................................................................................................................... 46
5.4 Defining PPIs ................................................................................................................................. 46
5.4.1 In general ............................................................................................................................................ 46
5.4.2 Large scale protein-protein interaction determination ...................................................................... 47
5.4.2.1 Yeast-Two-Hybrid and variants .................................................................................................. 47
5.4.2.2 Tandem affinity procedure (TAP) ............................................................................................... 48
5.4.2.3 Proximity labeling ....................................................................................................................... 49
5.5 Biophysical methods to confirm PPIs .............................................................................................. 52
5.5.1 Surface Plasmon resonance (SPR) ...................................................................................................... 52
5.5.2 Bio-Layer Interferometry (BLI) ............................................................................................................ 53
5.6 Protein-protein interactomes: Databases and Applications ............................................................. 54
5.6.1 Databases ........................................................................................................................................... 54
5.6.2 Validation of interactomes ................................................................................................................. 55
5.6.3 Analysis of interactome networks ...................................................................................................... 55
5.6.4 Some interactomes (no details) .......................................................................................................... 56
5.6.5 Some applications ............................................................................................................................... 57
5.6.6 The SARS-CoV-2 interactome (AP-MS studies) ................................................................................... 57
3
, CH 1: SAMPLE PREPARATION (KURT BOONEN)
1.1 PROTEOMICS
1.1.1 BOTTOM-UP APPROACH
The most commonly used proteomics technique is
bottom-up.
Proteins are digested into smaller peptides to identify
the peptides based on their fragmentation spectrum.
Proteins can be measured intact, but this is
significantly more difficult since the interpretation of
protein fragmentation spectra is hard. It is much
easier to interpret data from peptide analysis than
from complete proteins top-down approach, where
proteins are left intact: electrospray ionization (ESI) –
intact precursor ion – fragmentation.
A complex protein sample, results after digestion in a
complex peptide mix, is often separated by
electrophoresis or chromatography. Then, MS or
MS/MS analysis can be done on a simpler sample
(protein/peptide). Which is selected? Most abundant?
Think about this when conducting an experiment.
1.1.2 SHOTGUN PROTEOMICS
Shotgun proteomics acquire as much fragmentation spectra as possible from a sample and identify them! If the
MS software selects the peptides for fragmentation based on what is detected in MS1 we call it data dependent
acquisition (DDA). Shotgun is usually bottom-up.
After digestion, we have a
mixture – separated with LC –
then with MS1 see what is in it –
fragmentation.
Data-dependent acquisition
(DDA) = when the MS software
selects peptides for further
fragmentation based on what is
detected in MS1.
1
