Advanced Protein Technology and Proteome Analysis (UA_2079FBDBMW_2324)
Summary
Advanced Protein Technology and Proteome Analysis Summary (16/20)
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Module
Advanced Protein Technology and Proteome Analysis (UA_2079FBDBMW_2324)
Institution
Universiteit Antwerpen (UA)
This document relates to the course "Advanced Protein Technology and Proteome Analysis" within the Molecular Mechanisms of Diseases specialization in the first year of the master's program. The content of this document comprises a summary derived from the lecture slides presented by Professors Kurt...
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
4.3.2 MS versus NMR ................................................................................................................................... 34
4.4 LC-MS based metabolomics vs. proteomics..................................................................................... 34
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
5.6.2 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
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
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