Exam 2 Flashcards
If microarrays are easier, why use proteomics?
- It is proteins, not genes or mRNA, that are the functional agents of the genome
- Transcriptome information is only loosely related to protein levels: Abundant transcripts might be poorly translated, or quickly degraded; Hef1Alpha
Hef1Alpha
- hypoxia-inducing factor, expression higher in tumor than normal cells
- Lack of oxygen induces translation of protein. Protein transcript levels stay the same (transcript level can give us a good idea, but the translated protein is better)
- In presence of oxygen immediately gets degraded
Clinical proteomics- 2 ways to analyze presence of proteins
- Blood sample - use mass spec → proteomic image → pattern recognition, learning algorithm → early diagnosis of disease, early warning of toxicity
(Trying to find location of protein) - Tissue biopsy – use molecular-circuit image (see levels of proteins), → microarray chip → pattern recognition, learning algorithm → choose optimal therapy tailored to individual patient, monitor success of therapy
(Trying to find expression pattern of different proteins)
ELISA definition and purpose
- enzyme-linked immunosorbent assays
- Purpose: Detect presence of a specific protein in a liquid sample
Antibodies bind to RAS (target proteins) and fluores
(use antibody against RAS)
Antigen
Introduce antibody and immune system creates antibodies for protein X, collect those proteins with antibodies
Single protein analysis
begin with mixture of proteins
1. gel-based separation
2. sport excision (cut)
3. send peptides from a single protein for MS analysis
Shotgun
begin with mixture of proteins
1. digestion of protein mixture
2. LC peptides from many proteins for separation
3. MS for analysis
Protein sequence analysis definition
- Protein classification
- Helps characterize protein sequences in silico and allows prediction of protein structure and function
- Statistically significant BLAST hits usually signifies sequence homology
- Homologous sequences may or may not have the same function but will always have the same structural fold
3 steps to most proteomics experiments
- Preparation of a complex protein mixture
- Separation of protein mixture
- Characterization of proteins within mixture
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Separation techniques
2D gel electrophoresis description
Separation of proteins based on first isoelectric point and then molecular weight
proteins migrate through pH gradient until their overall charge is neutral
Polyacrylamide Gel Electrophoresis
- Protein separation based on size
- Small proteins migrate through gel matrix quickest
- Resulting gel has proteins separated
(Horizontally by IEP; Vertically by size/ molecular weight)
Blue spots represent proteins
2D gel electrophoresis limitations
Resolution
Representation
Sensitivity
Reproducibility
2D gel electrophoresis advantages
Established technology
* Still improving
* Quick
* Cheap (relatively)
Liquid chromatography description
- Proteins washed through capillary column (or columns)
- Separates based on: charge, Size, Hydrophobicity
- Depends on column matrix/eluent
- Usually 2 (or more) columns used
liquid chromatography limitations
expensive, difficult
liquid chromatography advantages
Resolution
Representation
Sensitivity
Reproducibility
Mass Spectroscopy description
- Analytical technique that measures Mass: Charge ratio (m/z) of ions
- 3 parts: ion source, mass analyzer, detector system
- Can Mass Spec whole proteins, but usually just peptides
- Samples always need in to be in gas/vapor form to get data
info given in MS analysis
- molecular weight
- number of specific elements (based on isotope peaks)
- molecular formula (with high resolution MS)
- reproducible fragment patterns (to get clues about functional groups and/or arrangement of components or to confirm compound identity)
3 parts of MS, 4 steps
1. Sample port- in vapor state; samples attacked by electron beam
2. Electron beam- attacks sample, e released from sample, makes each sample positively charged; m/z= +1, so sample is separated by mass
3. magnetic field= move in one direction toward detector
4. Detector : detects different fragment sizes based on MASS
what is X-Ray crystallography and what does it do?
- very high resolution microscopy to to visualize structure arrangement at the atomic level (helps understand protein function)
- beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions
- Produce 3D image of density of electrons from angles and intensities of the diffracted beams
- Useful to explore crystals because X-rays have wavelengths similar to the size of atoms
principle behind X-ray crystallography
X-rays are diffracted by crystals
X RAY DIFFRACTION
- uniformity of light diffraction of crystals to determine the structure of a molecule or atom
- X-ray beam “hits” the crystallized molecule
- The e- surrounding the molecule diffract as the X-rays hit them. This forms X-ray diffraction pattern
properties of X-ray
- EM radiations with a wavelength between 10A to 0.01A
- In Free Space, travel in straight line with a speed of 1,86,000 miles/sec (same as visible light)
- Cannot be heard, or seen, or Smelt
- Cannot be Reflected, Refracted or Deflected by magnetic or Electric Field
- Interference, Diffraction and Refraction similar to Visible light
- Produce an Electric field at right angles to their path of propagation
- Can penetrate liquids, solids and gases
- Interact with materials they penetrate and cause ionization
- Have a germicidal or bactericidal effect
- Can produce an image on a photographic film
Crystals
- Have vast number of precisely ordered, identical molecules.
- When fully formed, they are placed in a tiny glass tube or scooped up with a loop made of nylon, glass fiber, which is mounted in the X-ray apparatus, directly in the path of the X-ray beam
- The searing force of powerful X-ray beams can burn crystal through a hole, if crystal left too long in their path
- To minimize radiation damage, researchers flash-freeze their crystals in liquid nitrogen.
- 3D repeating arrays of precisely packed molecules (but can be different shapes like perfect cubes or long needles)
a lot of the same protein
why do we need a crystal?
- The diffraction from a single molecule would be too weak to be measurable. So we use an ordered 3D array of molecules (like Crystal) to magnify the signal. To get the final 3D structure
- Even a small protein crystal might contain a billion molecules.
- If internal order of the crystal is poor, then the X-rays will not be diffracted to high angles or high resolution and the data will not yield a detailed structure.
- If the crystal is well ordered, diffraction will be measurable at high angles or high resolution and a detailed structure should result.
- The X-rays are diffracted by the electrons in the structure and consequently the result of an X-ray experiment is a 3D map showing the distribution of electrons in the structure
- calculates the position of every atom in the crystallized molecule after intensity of each diffracted ray is put into computer
*** result: 3D digital image of the molecule. This image represents the physical and chemical properties of the substance and can be studied in intimate, atom-by-atom detail using sophisticated computer graphics software **
crystals MUST be
small in size (less than 1 mm)
PERFECT (no cracks, impurities, or inclusions like air bubbles)
crystal limitations
- Must have a single, robust (stable) sample, generally between 50—250 microns in size
- Optically sample should be clear
- Twinned samples can be handled with difficulty
- Data collection generally requires between 24 and 72 hours
Steps in structure determination
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X-ray crystallography in HIV
- Scientists determined the X-ray crystallographic structure of HIV protease, viral enzyme critical in HIV’s life cycle
- Pharmaceutical scientists hoped that by blocking this enzyme, they could prevent the virus from spreading in the body
- By feeding the structural information into a computer modeling program, they could use the model structure to determine the types of molecules that might block the enzyme
X-ray crystallography in dairy science
- Elucidation of compounds present in milk obtained through structure function relationship
- Stewart has shown that even solutions tend to assume an orderly arrangement of groups within the solution
- Hence, liquid milk show some type of arrangement
- The mineral constituent and lactose are the only true crystalline constituents in dairy products that can be analyzed by X-ray
X-ray crystallography uses
- To study materials that form crystals like salts, metals, minerals, semiconductors, as well as various inorganic, organic and biological molecules.
- Determine e-density (mean positions of the atoms in the crystal their chemical bonds)
- Size of atoms, the lengths and types of chemical bonds, and the atomic-scale differences among materials
- structure and function of many biological molecules, including vitamins, drugs, proteins and DNA
- Characterizing the atomic structure of new materials and in discerning materials that appear similar by other experiments.
- can account for unusual electronic or elastic properties of a material, shed light on chemical interactions and processes, or serve as the basis for designing pharmaceuticals against diseases.
what 2 jobs does DNA have?
- store info: Ensuring that information is passed on to each new cell upon division (and the next generation)
- direct the synthesis of proteins: necessary to carry out the functions of living organisms
“Sequencing” DNA
- the elucidation of the order of the bases in an organism’s DNA strand
- The unique order of your bases greatly influences your health
ex: What disease you are more - or less - prone to; How you will react to different medications
First gen sequencing
PCR, blots; old school; transcription level
RNA sequencing
averages data of all data. Doesnt explain difference between cells
Foreign vectors to clone genes
YAC: yeast artificial chromosome; used if cloning BIGGER genome
BACs: bacterial-based cloning system; used if cloning SMALLER genome; based on E. coli F factor (fertility plasmid): replication control; low copy number: low yield of DNA by standard methods; reasonably stable (small genome small plasmid)
contigs
- overlapping DNA segments that together represent a consensus region of DNA
- if smaller, can more easily find match
sanger sequences benefits
- fast, cost-effective for low numbers of targets (1-20 targets)
- familiar workflow
sanger sequences challenges
- low sensitivity (limit of detection approx 15-20%)
- low discovery power
- not as cost-effective for high numbers of targers (>20 targets)
- low scalability due to increasing sample input requirements
Targeted NGS benefits
- Higher sequencing depth enables higher sensitivity (down to 1%) to detect low-frequency variants
- Higher discovery power, mutation resolution, sample throughput
- More data produced with the same amount of input DNA
- Lower limit of detection
- Higher throughout with sample multiplexing
- Sequence 100-1000s of genes/regions simultaneously
sanger sequencing steps
- DNA is fragmented and cloned to a plasmid vector
- Cyclic sequencing reaction
- Separation by electrophoresis
- Readout with fluorescent tags
sanger sequencing definition
- 1st gen seq
- AKA dideoxy sequencing or chain termination
- Based on the use of 2’3’ ddNTP’s in addition to the normal nucleotides (NTP’s) found in DNA
how are ddnNTPs different from nucleotides?
ddnNTPs are essentially the same as nucleotides except they have a H on the 3’ carbon instead of OH
when happens when ddNTPs are integrated into a sequence?
- prevent the addition of further nucleotides
- occurs because phosphodiester bond cannot form between the ddNTP and the next incoming nucleotide, and thus the DNA chain is terminated
what happens to chain if:
there is no more addition of nucleotides?
add ddNTP
no more nt: chain will be terminated
ddNTP: chain elongation stops because do not allow to form phosphodiester bonds with new nt
Automated Sanger sequencing
the oligonucleotide primers can be
“endlabeled” with different color dyes, one for each ddNTP and fluoresce at different wave-length
Next gen sequencing
- mostly produce short reads from < 400 bp
- read numbers vary from 1 mill to 1 bill per run
- illumina, emPCR, bridge amplification, RNA seq, faoform
- 2 categories: sequencing by synthesis vs ligation
what are other names for next gen sequencing
- 2nd gen seq
- Massively parallel seq
- Ultra high-throughput seq
bridge amplification
- DNA molecules are repeatedly replicated on a glass flow cell containing complementary oligonucleotides.
- aka DNA cluster generation
clonal NGS
- emPCR
- bridge amplification (DNA cluster generation)
emulsion PCR definition
- dilution and compartmentalization of template molecules in water droplets in a water-in-oil emulsion
- each droplet has a single template molecule (bead) that binds to DNA molecules and functions as micro-PCR reactor
- avoids formation of unproductive chimeras between similar DNA sequences
- recovery of products involves repeated extraction with hazardous organic solvents followed by purification using silica-based columns (complicated)
emPCR steps
- generate emulsion by stirring
- generate clonal beads by emulsion PCR
- extract beads from emulsion (wash beads and do qPCR for yield)
- quantify yield by real time PCR
- flow cytometry analysis
magnetic beads, denature, vortex, separate template you prepared, sequencing
Bridge amplification procedure
1. Preparing genomic DNA sample: fragment DNA of interest into smaller strands that can be sequenced (sonication, nebulization, enzyme digestion), ligate adapters, denature dsDNA into ssDNA by heating to 95C
2. Attach DNA to surface- ssDNA is bound to inside surface of flow cell channels; Dense lawn of primer on the surface of the flow cell
3. Bridge amplification- Unlabeled nucleotides and polymerase enzyme are added to initiate the solid phase bridge amplification
4. Fragments become double stranded- shows the work done by the sequencing reagents: primers, nucleotides, polymerase enzymes, buffer
5. Denature the double stranded molecules- original strand is washed away, leaving only strands synthesizes to the oligos attached to the flow cell
In bridge amplification, what are adaptors complimentary to?
oligos
Illumina sequencing advantages
- proven base calling accuracy
- cost effective
- high quality pairwise alignments
- unbiased genome coverage
PACBIO sequencing
- smart cell aka ZMW (zero mode waveguide)
- detector detects as it is being synthesized, and every time a nt gets incorporated, it is detected and fluoroform falls off
- sequencing while synthesis
- uses different phospholinked hexaphosphate nucleotides
ChIP-seq
Combining DNA with motifs produces binding predictions for TFs
- measures TF binding but requires a matching antibody
- transcriptional regulation
- TFs block DNA cleavage and recognize motifs (signatures of DNA seq)
- (between hyper-sensitive sites, protected from any enzymatic digestion)
oxford nanopore
DNA sequenced by threading it through a microscopic pore in a membrane; NO SEQUENCING WHILE SYNTHESIZING, just reading the DNA; 2 proteins
- top unzips DNA helix into 2 strands
- creates pore in membrane and holds an “adapter molecule”
- flow of ions through pore creates a current. Each base blocks the flow to a different degree, altering the current
- adapter molecule keeps bases in place long enough for them to the identified electronically
genetic variation
1. point mutation (nonsynonymous vs. synonymous)
2. insertion/ deletions (indels)
3. copy number variations (CNVs)
4. structural variants (SV)- intrachromosomal (large indels, duplications, inversions, insertions, deletions)
interchromosomal (balanced and imbalanced translocations)
oconomic diagnostic mutation analysis
- rapid mutation for point-of-care analysis
- analysis of identified cancer drivers
- determination of pathogenic mutations
- ex: nonsense mutation in SMAD4
DNA methylation to histone protein
acetylation –> activate
DNA methylation to CG island
(in promoter) –> silences DNA
pyrosequencing principle
- non-electrophoretic sequencing-by-synthesis technique
- uses enzymatic system based on luciferase to monitor DNA production
- based on the detection of pyrophosphate (PPi when a nt is incorporated into a growing DNa strand during DNA synthesis)
- the incorporation of a nt triggers a cascade of enzymatic reactions that ultimately produce light, which is measured to indicate which nt was added
what can pyrosequencing help with?
- cDNA analysis, mutation detection
- re-sequencing of diseases linked genes
- viral and bacterial typing, and SNPs
pyrosequencing steps
- primer is hybridized to a SS, PCR amplified, DNA template…. and incubated with enzymes
- dNTP — DNA polymerase–> PPi release
- PPi –sulfurylase–> ATP + luciferin
- ATP + luciferin –luciferase–> oxyluciferin (LIGHT)
- light is detected by CCD camera and seen as a peak in the program. Each light signal is proportional to the number of nucleotides
- apyrase degrades unincorporated dNTPs and ATP to reset for next nt addition. When degradation is complete, another dNTP is added
pyrosequencing strengths
- delivers the “gold standard” of genetic analysis- the sequence itself
- fast (hands-on time: 1 hr; samples on machine: 10 mins) (ex: haemachromatosis mutations previously used PCR + restriction digest)
- quantification: important for mitochondrial mutations (previously used MS-PCR end-point PCR)
- genotyping straight-forward- pyrograms are easy to interpret
- machine + vacuum prep station require little maintenance
- can take pyrosequencing plate back through the vacuum prep station (add binding buffer to plate)
What is the light seen in pyrosequencing?
conversion of luciferin to oxyluciferin
in pyrosequencing, the intensity of light produced will be proportional to……..
the amount of ATP generated in reaction before, which is proportional to the amount of nucleotide added
In pyrosequencing, each nucleotide incorporation generates….
a characteristic light peak
Why are metabolomics important?
- can give instant snapshot of the physiology of cells
- functional end-point of physiology and pathophysiology
- direct mirror of environmental influences (Mal-nutrition, exercise, medication)
- metabolites are the ultimate result of cellular pathways
Why are metabolomics difficult?
- concentration of cellular metabolites vary over several orders of magnitude (mM to pM)
- differences in mw (20-2000 Da)
- high turnover rates
- some metabolites are labile
- requires that whatever chemical info it generates must be linked to both biochemical causes and physiological consequences (must combine bioinformatics and cheminformatics)
What can metabolomic assessment be pursued in?
in vitro
in vivo using cells, fluids, or tissues
biofluids are easies to work with: (serum, plasma, urine, ascitic fluid/pleural fluid, saliva, bronchial washes, prostatic secretions)
Warburg effect
tumor cells do glycolysis intead of ETC even in the presence of oxygen
metabolic profiling vs fingerprinting
profiling- quantitative study of a group of metabolites
fingerprinting- measures a subset of the whole profile with little differentiation or quantitation of metabolites
targeted vs untargeted metabolomics
targeted- known metabolites for specific pathways are targeted; measuring influence of therapeutics or genetic modifications on a specific enzyme
untargeted- measuring as many metabolites as possible (overall; at different stages of development) to generate a metabolic profile of a sample
Metabolomic samples and usage of metabolites
sample collection and handling
due to high susceptibility of metabolic pathways to exogenous environment, maintaining a low temp and consistent sample extracion is important
to make metabolomics fully integrated with omics, the data has to be…..
managed, stored, standardized
metabolic applications
- pharmacology & pre-clinical drug trials
- toxicology
- transplant monitoring
- new-born screening
- clinical chemistry
- tool for functional genomics
key limitation to metabolomics
the human metabolome is not at all well characterized
non-invasive detections for different cancers
prostate: sarcosine in urine
breast: choline
lung: breathe analysis
ovarian: separation rates
what is the goal of using metabolomic assessments for response to therapy?
to define pretreatment metabolic profile
metabolic detection of imatinib resistance
- decrease in mitochondrial glycose oxidation
- nonoxidative ribose synthesis from glucose
- highly elevated phosphocholine levels
example that not all metabolites can be identified
- carcinoma pancreas
- unresolved complex matter (UCM) much higher in those with cancer
pharmacometabolomics
detection of chemotoxicity using metabolomics (to predict individual variation in drug response phenotypes)
metabolomics data analysis steps
- Gather spectral data set for pattern recognition
- linking specific spectral region causing group clustering to specific metabolite based on its NMR chemical shifts
- quantitation & association of putative biomarkers with respect to particular characteristic or outcome
