week 6 Flashcards
1
Q
What is a proteome?
A
Entire complement of proteins (including post-translational modifications) present in a cell, tissue, organ or organism
2
Q
What is proteome derived from?
A
Protein and genome
3
Q
Define proteomics
A
large-scale study of a proteome using an array of proteomic methods
4
Q
what does metabolome provide?
A
biological status
5
Q
What are the data collected for proteomics?
A
- protein location
- abundance turnover
- Translational modifications
6
Q
what is an example of post-translational modification?
A
- hyperphosphorylation of tau in Alzheimer’s disease
7
Q
What do researchers use these 3 areas to study?
A
- protein activity
2. protein-protein interactions
8
Q
What can alter protein function?
A
- phosphorylation by inducing conformational changes
2. affecting protein-protein interactions
9
Q
What is phosphorylation state regulated by?
A
tightly concerted action of kinases and phosphatase
10
Q
What can proteomic approach be used to study?
A
- post-translational modification and their impact in human health and disease
11
Q
What are two major approaches of discovery phase?
A
- knowledge-based approach
2. unbiased approach
12
Q
What is the biomarker workflow divided into?
A
3 main parts:
- Discovery
- Verification
- Validation
13
Q
What is knowledge-based approach?
A
selection of biomarker candidate is based on existing molecular mechanism underlying disease initiation or progression
14
Q
What is unbiased approach?
A
untargeted identification of differentially expressed proteins between 2 analysed groups
15
Q
what does a small sample size at discovery phase lead to?
A
- overestimation of accuracy of biomarker performance and brings the reliability of findings into question
16
Q
What does the classical proteomics work flow include?
A
protein separation using gel based or gel free techniques followed by identification by mass spectrometry
17
Q
what can we look for in unbiased approach?
A
protein complement amongst 2 biological samples
compare and contrast and see what differs
18
Q
what are examples of a proteomic discovery methods?
A
- Gel electrophoresis (e.g. 2D-GE)
- Matrix-assisted laser desorption/ionisation (MALDI)
- surface-enhanced laser desorption/ionisation (SELDI)
- Isobaric protein tagging
19
Q
why is 2D-GE a widely-used method?
A
- high-resolution analysis of complex protein mixtures from a biological sample
20
Q
What does 2D-GE separate proteins by?
A
- Isoelectric point (pI)
2. Size (MW)
21
Q
Why may 2D-GE be an important method?
A
identifying between proteins that may be same molecular weight but have different isoelectric point
22
Q
What is the advantage of 2D-GE?
A
ability to resolve thousands of proteins in a single gel
23
Q
What is used to separate the proteins by isoelectric point?
A
- pH gradient
2. then the sample run via molecular weight (2D separation process)
24
Q
What were researchers able to do on computer base?
A
pinpoint and zoom in on certain protein to look at them for further analysis
25
How are proteins distinguished in a complex mixture?
1. proteins are separated in 2D isoelectric point and molecular weight
26
What is the disadvantage of 2D-GE?
1. moderate reproducibility
| 2. limited detection capacity
27
What are the proteins that do not appear on the gel?
1. Hydrophobic proteins
2. Low abundance proteins
3. Proteins that are above and below the pore size of gel
28
What is isoelectric focusing used to separate?
proteins in the first dimension
separates proteins based on their isoelectric point
29
What can isoelectric point be run in?
1. tube gels
| 2. Fixed gradient gel strips (IPG strips)
30
What does IPG strips have?
1. PH range - a researcher is able to select a specific PH range depending on their environment
31
What is PAGE (SDS-PAGE) used to separate?
proteins in the second dimension
this separates proteins on the basis of their size
32
What are examples of protein visualisation?
1. Coomassie blue (quick stain) - most common
2. Silver stain (sensitive stain)
3. fluorescently labelled proteins (e.g. DIGE)
33
What is a disadvantage of Fluorescently labelled protein?
It uses fluorescent scanner to distinguish proteins which is very expensive equipment
34
In pharmaceutical company, what stain is more favoured in labs?
1. Coomassie blue
| 2. Silver stain
35
What is gel scanning and computer software analysis?
1. add gel to scanner
| 2. computer software captures whats on the gel and analyse it based on how dense a protein spot is compared to another
36
What are examples of protein analysis?
1. comparison to master gels
2. Densitometry
3. Post translational modifications
37
Densitometry
increased and decreased proteins
38
Post-translational modification
1. computer software - see PTM
2. changes the properties of protein
3. alter position of spot
4. able to see if protein is phosphorylated or glycosylated
39
How are Gel spots excised and identified?
Mass spectrometry
40
What serves as a classical approach in analysis of differentially expressed proteins?
2D-GE followed by mass spectrometry
41
What is mass spectrometry?
analytical technique
samples are ionised into charged molecules
their mass-to-charge ratio is measured
42
What does a mass spectrometer have?
1. Ion source
2. Mass analyser
3. Ion detector
43
What does the component of mass spectrometer vary on?
1. Purpose of mass spectrometer
2. Type of data required
3. Physical properties of the sample
44
what is the principle of mass spectrometer?
1. a sample is added to a mass spec
2. ionized
3. Accelarated
4. undergoes electromagnetic detection
5. Detected
6. amplified into a signal
45
What does mass spec allow us to separate?
molecules based on mass-to-charge ratio
| which allow us to identify them
46
What is Matrix-assisted laser desorption/ionisation (MALDI)?
mass spectrometric technique used to identify and analyse biomolecules
47
What are examples of MALDI?
1. proteins
2. peptides
3. oligonucleotides lipids
4. metabolites
48
What is the process of MALDI?
1. The sample is mixed with a suitable matrix material and is applied to a metal plate
2. A pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material
3. Analyte molecules are ionised by being protonated or deprotonated in the hot plume of ablated gases
4. Accelarated into mass-spec to analyse them
49
What are the 2 main functions of matrix for MALDI?
1. Dilute and isolate protein peptide or general biomolecule
2. Acts as a mediator to absorb energy via the laser ionisation
3. Higher absorption capacity (protect sample from the laser ionisation)
50
What is the desorption process
1. The matrix completely detaches from the surface
| 2. Undergoes desolvation- removal of matrix and ionisation
51
Why is the matrix a key?
1. choose which analyte molecule you want to dilute
| 2. mediate energy absorption process
52
What are the 3 most commonly used for proteomics ?
1. 2, 6-dihyroxyacetophenone
2. alpha-cyano-4-hydroxycinnamic acid
3. 2,5-dihydroxybenzoic acid
53
What is key to the method of MALDI?
1. The matrix
| 2. Selection of matrix substances
54
What is surface-enhanced laser desorption/ionization (SELDI)?
1. A soft ionization method in mass spec
2. analysis of protein mixtures
3. Variation of MALDI
4. Proteins of interest in a sample become bound to a surface before MS analysis
55
What is SELDI used with?
1. Time-of-flight (TOF) mass spectrometers
| 2. used to detect proteins in tissue samples, blood, urine, or other clinical samples
56
What is a key principle of SELDI?
1. Sample is exposed to array chips with different active chromatographic surfaces that enrich certain groups of proteins
57
What is an example of a CHIP?
1. Q10
| 2. useful for detecting anions
58
What is key difference between MALDI and SELDI?
1. SELDI is able to use array chips to target certain groups of proteins not only via MW as in MALDI but target proteins that bind to metals
59
What does chromatographic surface arrays include?
1. Immobilized metal affinity capture (IMAC)
2. this targets proteins that bind to metal ions
3. binding of metal ions is seen in a number of neurodegenerative disease states
60
C10
Cation exchange chip
61
Q10
Anion exchange chip
62
What is Isobaric protein tagging?
1. Isotopic chemical labelling technique used for protein identification and quantification through mass spectrometry
63
What does Isobaric protein tagging allow?
1. Identification and quantifcation of labelled proteins in a specific sample within a mixture of samples
64
What is an advantage of Isobaric protein tagging?
Multiple samples can be run simultaneously and relative protein levels can be compared within a single experiment
1. cuts down the number of experiments performed and variations in experiments
65
What are the peptides labelled with Isobaric mass tags?
1. iTRAQ
| 2. TMT
66
What is TMT?
1. Tandem Mass Type
2. cannot be distinguished by the parent ion m/z
3. can be distinguished upon fragmentation
67
What is the structure of Isobaric mass tags?
Has:
1. Reporter group
2. Cleavable linker
3. Mass normalisation groups
4. Reactive groups
68
What is a reporter group?
1. Reports the abundance of a peptide upon MS/MS from individual samples in a mixture
69
What is a cleavable linker?
enables release of TMT report fragment from the whole tag upon MS/MS
70
What is mass normalisation group?
balances mass differences from TMT reporter fragments to ensure the same overall mass of the label reagent
71
What is reactive group?
Allows for labelling of amino groups (N terminal and internal lysines)
72
What does each tag have?
Different isotope configuration between reporter group and mass normalisation group
73
What is methodology of Isobaric protein tagging?
1. 6 protein samples are reduced via alkylation which prevents disulphide bridges forming
2. They are digested and labelled with tandem mass tags
74
What does Isobaric protein tagging allow?
1. Mix samples
2. Purify
3. Perform mass spec
75
What is seen upon fragmentation?
1. Isotopic distribution of these different types
76
What does the verification phase of a proteomic workflow involve?
1. confirmation of candidates abundance and presence in a clinical sample
77
What happens in the discovery phase?
1. unbiased or untargeted approaches are applied to define a candidate or a set of candidate biomarkers
78
What does the verification phase represent?
1. targeted approach for further evaluation of biomarker's sensitivity, specifity and predictive capabilities
79
Where are verification studies performed on?
appropriate biological specimen that may be further used in clinical practice
80
What does the verification studies require?
larger patient populations than the discovery phase
81
What does the validation phase of a proteomic workflow involve?
Evaluation of clinical utility and analytical performance
82
Where does the validation phase have to be performed in?
1. Independent sufficiently large sample set reflecting the heterogeneity of targeted population
83
What does the validation require?
1. Quantitative
2. Robust
3. High throughput methodology
84
What underlies the verification and validation phase?
1. large samples can be analysed in a quick and easy manner
85
What is considered as the gold standard in clinical application?
antibody-based approaches
86
What are examples of proteomic verification and validation methods?
1. Multiple reaction monitoring (MRM)
2. Western blotting (Immunoblotting)
3. Enzyme-linked immunosorbent assay (ELISA)
4. Multiplex assays (e.g. Luminex)
87
What is Multiple reaction monitoring (MRM)?
Mass spectrometric technique used for highly specific and sensitive quantitative measurement of target proteins
88
What MRM specify?
1. parent mass of the protein for fragmentation
| 2. Delivers a unique fragment ion that can be monitored and quantified
89
What results in a single reaction monitoring (SRM)?
Fragment ion counted over time
90
What are we able to target in MRM?
1. multiple peptides
2. multiple protein
in the same experiment
91
What does MRM use?
1. Triple quadruple mass spectrometery
92
What does the triple quadruple mass spectrometry do?
1. Targets the parent ion
2. target the fragmented product ions
3. increases specifity and sensitivity
93
What are the 3 main stages of MRM?
1. peptide selection
2. fragmentation
3. fragment selection
94
What is peptide selection?
parent ion is pre-selected with a specific m/z with mass filter quadruple Q1
95
What is Fragmentation?
Parent ion is induced to fragment in the collision cell quadruple Q2
96
What is Fragment selection?
Product ions are mass analysed using third Quadruple 3
97
What is western blotting?
1. Immunological technique
2. Detect a protein of interest from complex mixture
3. using enzymatic or fluorescently labelled antibodies
98
What is the first stage of western blotting?
1. Gel electrophoresis
| 2. Separates native or denatured proteins by length of polypeptide/basis of size
99
What is the second stage of Western blotting?
1. Proteins are transferred to a membrane ( nitrocellulose or PVDF) - Transfer process
100
What is the 3rd stage of Western blotting?
1. Proteins are probed using antibodies specific to the target protein
2. protein-antibody tagging can be detected
101
What are examples of protein-antibody binding?
1. Colorimetric
2. Chemiluminensce
3. Fluorescence
102
What does Western blotting use?
1. specific primary antibody to peptide/protein of interest
| 2. uses a secondary antibody which is labelled for substrate detection
103
What are the limitations of western blotting?
1. It requires a specific antibody
2. High cost (e.g. reagents, equipment)
3. High technical demand (optimisation required)
4. Transfer efficiency (<10 kDa proteins may not be retained by the membrane)
5. High background (non-specific binding)
104
What are the advantages of western blotting?
1. High sensitivity (picograms to nanograms)
| 2. High specifity (e.g. separation by size, charge, conformation and by antibody-antigen interaction)
105
What is ELISA?
1. plate-based immunological technique used for quantifying substances such as proteins, peptides and antibodies
106
What are different forms of ELISA?
1. Direct ELISA
| 2. Indirect ELISA
107
What is Direct ELISA?
1. antigen is bound to well of plate
2. a primary antibody is labelled
3. Substrate is added and we see a colour change on this interaction
4. just uses primary labelled antibody
108
What is Indirect ELISA?
involves 2 binding process of primary antibody and labelled secondary antibody
1. The primary antibody is incubated with the antigen followed by incubation with the secondary antibody
109
What is Sandwich ELISA?
1. Sensitive and robust method which measures the antigen concentration in an unknown sample
2. The antigen of interest is quantified between 2 layers of antibodies: the capture and the detection antibody
110
What are the advantages of ELISA?
1. Quantitative (concentration of antigen in sample)
2. High throughput (e.g. commercial kits available)
3. Time efficent and easy to perform
4. cost-effective
111
What are limitations of ELISA?
1. Limited antigen information (i.e. presence or absence of protein in sample only)
2. Temporary read outs (i.e. short time-span of enzymatic reactions)
3. Unable to provide a simultaneous quantitative analysis of multiple potential biomarkers
112
What is Digital Elisa (e.g. Simoa)?
1. single molecule array
| 2. Bead based technology that uses digitial counting to increase senstivity compared to a traditional ELISA
113
What does the digital approach make use of?
1. arrays of femtoliter-sized reaction chambers that can isolate and detect a single immunocomplex
114
What does the digitial ELISA use?
beads that can use amino-counting
115
What is Multiplex assay?
e. g. Luminex
1. Immunoassay based on priniciples of ELISA but instead uses magnetic beads to simultaneously measure multiple analytes in a single experiment
116
What does Multiplex assay uses?
1. Technology that uses up to 100 colour-coded bead sets
117
What can be generated in Multiplex assay that can be spectrally distinguished?
changing relative amounts of 2 dyes
1. one hundred unique sets of microsphere beads are generated
118
For multiplex assay, what can the ends of each beads be conjugated with?
Different specific reactant (e.g. antibodies)
| and up to 100 different species (e.g. protein) can be simultaneously measured in a single tube or microplate well
119
What is the first step of Multiplex assay?
1. colour coded beads (pre-coated with a specific capture antibodies) are added
120
What is the second step of Multiplex assay?
2. antibodies captures the analyte of interest
Biotinylated detection antibodies specific to the analyte of interest are added and form an antibody-antigen sandwich
121
What is step 3 of Multiplex assay?
3. Phycoerythrin (PE) conjugated streptavidin is added
122
What is step 4 of Multiplex assay?
4. Beads are read on a dual-laser flow-based detection instrument
one laser classifies the bead and determines the anaylte that is being detected
the second laser determines the magnitude of the PE-derived signal, which is in direct proportion to amount of bound analyte
123
What is an example of proteomic workflow?
Alzheimer's disease
124
Alzheimers disease
1. Reported by Alois Alzheimer in 1906 in patient who suffered impaired memory, disorientation, inability to use langage and ''psychosocial incompetence''
125
What was described in the post-autopsy of Alzheimers?
A peculiar severe disease process of the cerebral cortex
there are plaques between neurons and tangles within neurons
126
How is plaques formed?
protein amyloid
127
How are tangles formed?
protein tau
128
What is deposited first that spreads as disease progresses?
Amyloid plaques
129
What leads to neuronal cell death?
Amyloid and tau
130
What are the present limitations of pathological analysis?
1. Aggregates are revealed only by post-mortem analysis
2. Earliest changes in disease are missed
3. End-stage tissue is depleted of neurons
4. Post-mortem delay affects proteins
5. small number of accumulating proteins revealed
131
What is Alzheimer's disease diagnosed by?
1. Medical history
2. Clinical examination
3. Cogntive examination
4. Neuropsychological examination
5. Diagnosis of exclusion
132
What overlaps the symptoms of Alzheimer's disease?
Vitamin B12 deficiency
133
What was the aim of the study?
Identify diagnostic biomarkers For AD in peripheral venous blood using a number of proteomic approaches
134
What 2 parts was the study broken down into?
1. Discovery phase
| 2. Validation phase
135
What requires a larger population size?
Validation phase
136
Discovery phase cohort selection
1. 50 people with AD were recruited through secondary care services
2. 50 normal elderly controls were also recruited through primary care services
137
Validation phase cohort selection
1. 511 subjects with AD and normal elederly controls were recruited
138
What is described as a true validation phase?
1. cohort size would need to be increased
| 2. fully quantitative assays would need to be implemented
139
Why was blood chosen as the biological sample for the proteomic platform?
1. cost effective
2. Less Invasive
3. Routine collection
140
What was absorbed into blood each day?
| rich source of biomarkers
~500ml of CSF
141
How was blood collected?
1. Anti-coagulated tubes (Na-EDTA)
| 2. Serum tubes
142
What is important to process blood very quickly?
Blood samples were centrifuges at 3000rpm (10 mins at 4 degrees) within 2 hours of collection
143
What is two-dimensional gel electrophoresis (2D-GE) used for?
1. plasma profiles of the discovery phase cohort
144
What is isoelectric focusing used to separate?
proteins in the first dimension (IPG strips PH 3-10)
145
What is SDS-PAGE (10% gel) used to do?
Separate proteins in the second dimension
146
What was done in both case and control groups?
Gels were silver stained (plus one silver staining kit) and analysed quantitatively (Melanie 2D software) to identify spots (proteins)
147
What was the results for 2D:GE?
used computer based software - able to distinguish proteins on basis of isoelectricc point and MW
a total of 217 proteins spots identified that matched in cases and controls
148
How was the mean of integrated optical density of each protein assessed?
non-parametric test
e.g. Mann-Whitney U test
149
What was false discovery rate index used for?
computed and 15 proteins spots were identified to have a FDR < 0.50
150
What was significantly different between both groups?
15 proteins in total
151
what happened to the 15 protein spots?
Excised and identified by liquid chromatography tandem mass spectrometry (LC/MS/MS)
