Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

Techniques of Molecular Medicine > Proteomics > Flashcards

Proteomics Flashcards

1
Q

What does omics refer to?

A

Informally refers to genomics, proteomics or metabolomics

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is -ome used for?

A

To address the objects of study of such field, such as the genome, proteome or metabolome respectively.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are the aims of omics?

A

The collective characterization and quantification of pools of biological molcules that translate into the structure, function and dynamics of an organism or organisms

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is the proteome?

A

All the proteins expressed at any one time

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

In which aspect relevant to omic technologies are proteins and DNA different?

A

DNA is specific to a certain species or individuals

Proteins can be specific at many levels - population, person, tissue, cell, organelle

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What information can you get from the genome?

A

Functional information only

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What information can you get from the proteome?

A

How much there is

Activated or not

State of activation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

How much variation is there in the genome?

A

Little compared to the proteome

Arises only from the combinations of 4 nucleotides

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How much variation is there in the proteome?

A

A lot

Arises from combination of up to 20 amino acids

Increased further by the post-transcriptional modification

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

How many genes are there in the genome?

A

Around 25 000

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How many proteins are there in the proteome?

A

More than 1 million

Modification and splice variants increase the amount

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

The genome encompasses a —- code, whereas the proteomic code is highly —-

A

Static

Dynamic

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What are prepro regions?

A

Proteins are often made as precursors or pro-proteins

Not active until they have undergone some form of post translational modification

They also contain a signalling peptide (pre-pro peptide) to signal the cell whether or not they should be secreted or incorporates into the plasma membrane

This is a cleavable peptide that must be removed before the protein is activated

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What are examples of post translational modification?

A

Posphorylation

Acylation

Methylation

Ubiquitination

Glycolysation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Give an example where proteomics was used clinically

A

Ovarian cancer patients

Bioinformatics was combined with proteomics

This showed clusters identifying key genes involved, including Ubiquitin C and FGF4

Also showed gene outliers, which could be specific to the type of cancer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Which gene is specific and used clinically to diagnose ovarian cancer?

A

Mucin16

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What type of field is bioinformatics?

A

Very multidisciplinary

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What professions are involved in bioinformatics?

A

Biologists

Computer scientists

Mathematicians

Chemists

Statisticians

Physicists

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What does bioinformatics entail?

A

The study of biological information with a computer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What information can bioinformatics provide us with regarding the proteome?

A

Structural

Find networks

Quantify a protein

Generate models: how changes in environment can affect protein modulation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What 3 tools can be used for bioinformatics?

A

BLAST

GO

Panther

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Describe the BLAST bioinformatic tool

A

Takes a sequence and searches it

Compares the sequence to sequences in the database

Can be a protein or a gene

Uses this to guess the protein it is

Uses the conserved region of a protein to accurately predict its function

Can also give you the phylogeny information regarding that protein

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Describe the GO bioinformatic tool

A

Gene ontology

Associates the function of a protein with a word

Creates a library of these proteins regarding their functions

Clusters proteins with similar functions together

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

Describe the Panther bioinformatic tool

A

Looks at a list of proteins described by GO and returns the average most enriched processes

Cancer cells: apoptotic processes, immune cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Give an example of bioinformatic analysis done on a patient cohort
Asthma patients Grouped patients into eosinophilic, neutrophilic and non-allergic Showed that neutrophilic asthma formed 2-3 subgroups, where the effect of treatment and disease type were different Identified groups which shared genes
26
What, in terms of hypotheses testing, are omic technologies useful for?
Omics are good for generating hypotheses
27
What are prescriptive vs non-prescriptive approaches in proteomics?
Prescriptive approaches have defined targets Non-prescriptive approaches have unknown targets
28
What type of approach is used in protein arrays?
Prescriptive Used to identify a predefined set of proteins
29
What technique are protein arrays based on?
Antibody recognition
30
Are protein arrays classified as omic technologies?
Not yet We don't have all the antibodies for all the proteins Almost omic for yeasts
31
What type of approach is used in mass sectromertry ?
Non-prescriptive Identifies all the detectable proteins in a sample
32
What are mass spectrometry results based on?
Peptide m/z values
33
Are mass spectrometers classified as omic technologies?
Yes
34
What are the two proteomic technologies used?
Protein arrays Mass spectrometry
35
What is proteomic characterisation?
The characterisation and quantification of all polypeptide components present in a biological compartment
36
What are the three levels by which proteins are characterised?
Primary Secondary and tertiary Quarternary
37
What is the primary structure of proteins?
Amino acid sequence
38
What is the quarternary structure of proteins?
Protein-protein interactions Interactome
39
What are the questioned addressed by proteomics?
Which proteins are translated from the genome/transcriptome? Where are the proteins found? Which proteins do the proteins bind to? Are the proteins post-transcriptionally modified? Does the protein concentration/localization/ interaction/ modification differ between samples?
40
What are the pros of mRNA profiling?
Fairly omic Quick Relatively cheap
41
What are the pros of proteomics?
Good for looking at ECM and fluids such as CSF, plasma, serum and urine More informative and more accurate to focus on the active entities rather than their biosynthetic template to asses abundance, modification, turnover and localisation
42
What are the cons of mRNA profiling?
Bad at looking at certain ECM and fluids
43
What are the cons of proteomics?
Difficult to perform Expensive Not truly comprehensive
44
Do proteomes and mRNA profiles always correlate?
Some correlate well, some don't
45
What are the two reasons for why we need sample fractionation?
Dynamic range Complexity
46
Why does the dynamic range of a sample mean we need sample fractionation?
Some molecules very abundant in the serum This is too large for a single detector
47
Why does the complexity of a sample mean we need sample fractionation?
A large number of protein sequences 1 000 000 protein sequences This is too large for a single platform
48
What does proteomic mass spectrometry analyse?
Peptides produced by proteolytic digestion of samples
49
What important step is crucial to perform before fractionation?
Targeted depletion of abundant proteins So they don't interfere with results Immunoaffinity and ligand binding
50
What are the two types of non-targeted fractionation?
Protein fractionation Peptide fractionation
51
Two types of protein fractionation
1D or 2D electrophoresis 1D or 2D-liquid chromatography
52
Two types of peptide fractionation
Solution phase isoelectric focusing 2D HPLC
53
What is the key in fractionation?
Minimise experimental error
54
What are two ways to minimise the experimental error in fractionation?
High reproducibility Avoid non-specific losses
55
What are the two types of variation that exist in experiments?
Technical variation Biological variation
56
What is technical variation?
Experimental error Occur whenever we prepare or analyze samples
57
What are ways to reduce technical variation?
Standard operating procedures Quantifying using technical replicates
58
What are sources of biological variation?
Sample fidelity: are there components of your sample you are not aware of? Anatomical heterogeneity Longitudonal variation between periods of time Individual variation between patients
59
What are ways to reduce biological variation?
Increase cohort size Stratification - cluster variation Quantify using biological variations
60
Biological samples are often impure TRUE or FALSE
TRUE Due to contamination and cell and tissue heterogeneity
61
What are the reasons we need validation?
Variation (biological and technical) Proteomic results are based on probability Important identifications require validations using orthogonal techniques
62
What does validation due?
Make sure the identity of the sample is correct and it localises to the tissue/cell/organelle
63
What are orthogonal techniques?
Completely different technique than the one used in the experiment
64
What are examples of orthogonal techniques?
Immunohistochemistry ELISA Labelling
65
Why are mass spectometers used over ELISAS and protein blocks?
ELISAs and protein blocks look at a protein at a time Mass spectometers looks at many at the same time These techniques can be applied to lipid and metabolomics as well
66
What are the steps of mass spectometers?
Preparation Mass-spectometer Protein identification Analysis/bioinformatics Validation
67
Describe the preparation steps taken before the sample is inserted into the mass spectometer
Proteins collected from urine tissue, lung lavage ... Reduce proteins through alkylation, which breaks the disulfide bonds between cysteine and then stop the bonds from forming Digest using trypsin, which digests the C terminal of arginine and lysine Extract Label and pool Fractionate
68
Give an example of a validation technique
Identify a biomarker for ovarian cancer Look at serum of patients and compare it to healthy controls Then validate these using orthognal techniques
69
Why do we need to digest the proteins?
In order to be able to fragment and ionise them
70
Can we identify a protein using its ionised form?
No
71
Can we identify a protein using its fragmented form?
No
72
Can we identify a protein using the ionised form of its peptide?
No
73
Can we identify a protein using the fragmented form of its peptide?
Yes
74
What is the challenge with ionising proteins?
Very high energy needed for direct desorption and ionisation
75
What is the challenge with fragmenting proteins?
Difficult to produced ordered series of daughter ions producing any amino acid sequence information
76
What is required in order to ionise peptides?
High energy
77
What is the usefulness of the fragmented peptides?
Peptide fragmentation data can be interpreted to deduce amino acid sequence information
78
What are we measuring in a mass spectrometer?
The mass to charge ratio of peptides fragmented from the proteins we want to analyse
79
Describe how liquid chromatography works in collaboration with mass spectometry
Start off with complicated sample Separation technique is used, usually liquid chromatography Break down proteins into peptides Peptides are ionised through soft ionisation Peptides get pushed into the mass spectometer Mass spectometer measures the mass/charge ratio Take peptide and push it through another mass spectrum following further fragmentation Another spectrum is obtained, which helps identify what the peptide is and therefore quantify it
80
What does the mass filter do?
Guides the ions straight into the mass spec
81
What is the purpose of the separation techniques in mass specs?
Prevents the molecules from clustering together Separates them by size or chemical properties
82
Why do we break proteins down into peptides in mass spec?
They are more stable Can identify the peptides, which is used to quantify and identify whole proteins
83
What charge do proteins have following soft ionisation?
Positive charge
84
What is the benefit of the positive charge of proteins?
Helps filter out other substances
85
How do mass specs measure the mass to charge ratio of peptides?
Peptides are pushed into a vacuum The time taken for the peptide to travel through is measured We know the force needed to push the peptide through the vacuum, and the time So we use this to calculate the mass
86
Singularly charged peptides moves in vacuums with half the force as peptides with double charges TRUE or FALSE
TRUE
87
What are two techniques of soft ionisation?
Matrix-assisted laser desorption ionisation Electrospray ionisation with water
88
Describe the process behind MALDI
Energy from a laser goes into the matrix The matrix absorbs the energy and ionises some of the molecules Time of flight is then measured
89
What does the time of flight in MALDI represent?
Amount of time taken for the ionised molecule to hit detector on the back
90
What are the two techniques used in ESI?
Quadrupole Orbitrap
91
Describe the components of the quadrupole
Contains 4 poles 2 positive and 2 negative
92
How does a quadrupole work in identifying peptides?
The voltages are changed so that only one specific mass to charge ratio get through The other ones crash and burn on the side The positive rods remove molecules below a certain value The negative rods remove molecules above a certain value
93
Describe the structure of the orbitrap
Outer barrel-like electrode Coaxial inner spindle-like electrode that traps ions in an orbital motion around the spindle
94
How does the orbitrap work?
Ions are trapped because their electrostatic attraction to the inner electrode is balance by their inertia
95
What are the two types of mass spectra obtained following fragmentation?
Single stage MS Tandem MS
96
What information is obtained from single stage MS?
Protein mass data, including purity and integrity Peptide mass data using the fingerprint
97
What is the use of single stage MS?
Proteins are digested Mass spectra are formed, and every possible protein in the human proteome is digested and fingerprints are made We can then match protein samples to these fingerprints
98
What is the use of tandem MS?
We can measure the mass of the peptides Can put a particular peptide and run it through a collision cell to fragment it in a predictable way Then you measure the mass to charge ratios of all the fragment ions We use these ions to measure the sequence
99
What charge do tryptic peptides possess normally?
Double
100
What types of ions are produced from the ionisation of tryptic peptides?
Single charged b- and y- ions
101
Where are the b-ions retained?
In the N-terminal fragment
102
Where are the y-ions retained?
In the C-terminal fragment
103
What are 3 ways of quantifying peptides?
SILAC Label free Chemical mass tagging
104
What is SILAC?
Stable isotope labelling of amino acids in culture Gold standard
105
How is SILAC carried out?
Grow cells in heavy medium
106
What is the disadvantage with SILAC?
Does't work in animal or human experiments Too fast or not possible
107
Examples of label free quantification mechanisms
iBAQ Top3 EmPAI
108
What is the advantage of label free quantification mechanisms?
Does not modify proteins, so no risk of affecting ionisation or properites
109
What is the disadvantage of label free quantification mechanisms?
Have to do one sample at a time
110
What are examples of chemical mass tagging used to quantify proteins?
TMT iTRAQ
111
Describe the mechanism behind chemical mass tagging
Process of chemically modifying peptides according to their mass
112
What is the advantage of chemical mass tagging?
You can have different weighted tags which makes it high throughput
113
What is the most ideal quantification technique?
There is no one ideal single method Most use a combination of different methods

Techniques of Molecular Medicine (12 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions