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Samenvatting en aantekeningen Bioanalytical and Omit Techniques
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Bioanalytical & Omics TechniquesInhoud
Lecture 1: Genomics/transcriptomics/Chip-2-chip – Dr. Kuipers, 7 January 2022 .......................... 2
Functional Genomics.......................................................................................................................... 2
ChIP-on-chip: get an eye to the regulator .......................................................................................... 5
Lecture 2: Protein secretion & paper Cao exc. – Prof. Kuipers, Tuesday 8 February 2022 ........... 11
Functional genomics for analysis of protein secretion in Bacilli – molecular genetics ............... 11
Proteomics & molecular biology: verification of predictions.............................................................. 13
Lecture 3: Interactomics/PPI’s – introduction & review – Dr. Thunissen, Tuesday 10 February
2022...................................................................................................................................................... 15
Why study PPI’s? .............................................................................................................................. 15
Quick overview of experimental techniques used for discovering PPIs - Methods to identify PPIs
........................................................................................................................................................... 18
Lecture 4: PPI – Methods targeting co-complex methods – Dr. Thunissen, Friday 11 February
2022...................................................................................................................................................... 20
Tutorial 1 – Discussion of scientific papers – Dr. Thunissen. Friday 11 February 2022 ................ 27
Lecture 5: PPIs – Methods targeting binary PPIs / fluorescence-based methods – Dr. Thunissen,
Monday 14 February 2022 .................................................................................................................. 29
Lecture 6: Metabolomics/mass spectrometry - identification, quantification, metabolite analysis –
Dr. Permentier, Monday 14 February 2022 ....................................................................................... 36
Analytical techniques in Proteomics & Metabolomics ................................................................... 36
Mass spectrometry ........................................................................................................................... 43
Verschillende mass analyzers ......................................................................................................... 46
Lecture 7: Metabolomics/mass spectrometry - identification, quantification, metabolite analysis –
Dr. Permentier, Tuesday 15 February 2022....................................................................................... 52
Interpretation mass spectra MS ...................................................................................................... 52
Ionization techniques → ESI & MALDI ............................................................................................ 54
Liquid chromatography & LC-MS ..................................................................................................... 59
Fragmentation of ions by MS/MS, tandem mass spectrometry ................................................... 62
Peptide fragmentation & identification spectra used .................................................................... 65
Lecture 8: Metabolomics/mass spectrometry – identification, metabolite analysis – Dr.
Permentier, Tuesday 15 February 2022............................................................................................. 67
Protein identification with MS & database searching .................................................................... 67
Protein identification by searching in mass libraries ..................................................................... 72
Protein (peptide) identification ........................................................................................................ 74
MS-based metabolomics ................................................................................................................. 78
Lecture 9: Computional mass spectrometry and bioanalysis – Prof, Horvatovich, Thursday 17
February 2022 ...................................................................................................................................... 80
Introduction to proteomics bioinformatics...................................................................................... 80
Proteomics applications for Personalised Medicine ...................................................................... 88
1
©C.A.M. Mittendorff
,Lecture 1: Genomics/transcriptomics/Chip-2-chip – Dr. Kuipers, 7 January 2022
Examen over alle 4 papers & alle lectures
Vanmiddag: paper lezen: Boosting heterogous protein production yield by adjusting global
nitrogen and carbon metabolic regulatory networks in Bacillus subtilis
• Weak points in reviews
• Techniques & technologies used
• Etc. → zie nestor
Functional Genomics
• >500,000 genomes sequenced
• > 10% genes with unknown function
• Incomplete knowledge about:
o Cell physiology
o Diversity within a species
o Complex regulatory networks
o Macromolecular interactions
• Genomics, Transcriptomics, Proteomics, Metabolomics, Interactomics, Bioinformatics,
Doel: Whole cell physiology begrip
o Functional genomics is combinatie van transcriptome, proteome en metabolome
etc.
Required approach
• High troughput technologies → start door technologiën high troughput te maken rond
2000
o Use of robots
o DNA-microarrays/scanners
o 2D-electrophoresis
2
©C.A.M. Mittendorff
, o Gel-free analysis
o MALDI-TOF-MS, MS-MS
o Algoritms/software tools for
▪ Genome analysis/comparison
▪ Statistics
▪ Visualization, pattern recognition
▪ Structure/function/interaction prediction
• Moeilijk om high troughput te maken
▪ Whole cell simulations
o Structure/function determinations
o Moleculair interactions, kinetics
▪ Moeilijk om high troughput te maken
Analysis of gene expression by DNA-microarrays
Transcript profiling
In jaren 90 vergelijking van wild type vs. mutant/overexpression/other growth conditions etc. →
Analysis of gene expression by DNA-microarrays.
Totaal RNA werd geïsoleerd en gelabeld om cDNA te maken. cDNA was fluorescent gemaakt.
De mate van de kleur gaf aan in welke mate van hoeveelheid het cDNA aanwezig was.
Relatieve quantificatie, geen absolute quantificatie.
Limitations/problems with transcript profiling/DNA-microarrays
• Standardization, validation
• No direct correlation with proteome
o Regulatie in zowel transcriptie en translatie. Er is dus geen directe correlatie, wel
enige relatie.
• Significance levels of data
• Sensitivity (material availability)
• Lack of visualization software
• Costs (initial investments)
Let op! Tegenwoordig wordt transcriptomics vnl, gedaan door RNAseq → more expensive.
Research areas where DMA’s can be used
• Comparative genomics
o Genomotyping
o Architecture of gene regulatory networks
o Intergenic region arrays for protein interaction
• General/medical
o Link phenotype to gene expression profile
o Identify drug targets/efficacy of drugs
o Assess genetic polymorphisms
• - Microbial ecology
o Identification of pathogens
o Monitoring population dynamics
• Virtual cells
o Integration of transcriptome, proteome,
o Metabolome, interactions, visualization tools etc.
RNA quality control
Altijd belangrijk ook voor RNA-seq.
Pieken van ribosomaal DNA kunnen erg groot zijn en de pieken van mRNA laten
‘ondersneuvelen’. → Kijken naar verhoudingen voor kwaliteit controle.
→ Details zie ppt.
Case study transcriptomics
3
©C.A.M. Mittendorff
, Doel: finding a binding motif for CodY repression in L. lactis.
CodY-activity
DMA’s = DNA Micro Arrays
Oligopeptiden worden opgenomen, bijvoorbeeld via Opp.
CodY meet hoeveelheid free brange change aminoacids (free brange chain accumalutation in the
cell). CodY werkt als een ‘thermostaat’.
Bij veel vrije brange chain aminozuren die accumuleren in de cel → binding aan CodY. CodY wordt
dan werkzaam als een respressor voor genen van peptidases/proteases/uptake systemen. Door
downregulatie van deze genen → minder van deze proteinsystemen. Dus minder opname van
peptides etc.
CodY kan respressie geven
Op een gegeven moment is er een te kort aan free brange chain aminozuren. → Cody zal affiniteit
verliezen en zal van het DNA afvallen, dus geen respressie meer.
Identification shared motif in CodY regulated genes
Onderzoeksvragen & strategie:
Which genes are regulated by CodY?
No recognition site known for CodY; can we identify any?
Strategy: i) Create dataset of promoter sequences of hits of DNA microarray combine with
known targets
ii) Use MEME-software to identify motifs in common
iii) Use obtained motif to build weight matrix
iv) Visualize in G2D & predict novel targets
Transcriptome MG1363 WT vs. CodY
MG1363 is dezelfde strain maar dan zonder CodY-gen.
Aminozuren met nitrogen worden gereguleerd door CodY
Via deze DNA-micro-array-analyse geeft dataset of protomor sequences of hits of array → er is
een patroon.
→ Zie ppt. 24
Positie 3, 7 en 13 blijven altijd hetzelfde.
Search of motif in L. lactis MG1363-genome
Hoe lager in score hoe meer verschillen in score.
Mismatches kunnen een functie hebben in de cel. Bij een perfect motif
→ CodY bindt erg sterk. → Sterke regulatie.
Perfecte regulatie is niet altijd gewenst, liefst in een low level.
Minder sterke binding geeft minder sterke regulatie en dus gene expression.
EMSA
EMSA: The electrophoretic mobility shift assay (EMSA), one of the most sensitive methods for
studying the DNA-binding properties of a protein, can be used to deduce the binding parameters
and relative affinities of a protein for one or more DNA sites or for comparing the affinities of
different proteins for the same sites
His6-CodY binding to predicted targets
Electrophoretic Mobility Shift Assay → EMSA
• Radioactief gelabeld probe DNA
o Bevat de plaatsen waar CodY kan binden
• Shift naar boven omdat het complex bindt aan het DNA → complex heeft namelijk een
hoger MW
4
©C.A.M. Mittendorff
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