Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

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
Q

Give an example of bioinformatic analysis done on a patient cohort

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

What, in terms of hypotheses testing, are omic technologies useful for?

A

Omics are good for generating hypotheses

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

What are prescriptive vs non-prescriptive approaches in proteomics?

A

Prescriptive approaches have defined targets

Non-prescriptive approaches have unknown targets

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

What type of approach is used in protein arrays?

A

Prescriptive

Used to identify a predefined set of proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

What technique are protein arrays based on?

A

Antibody recognition

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

Are protein arrays classified as omic technologies?

A

Not yet

We don’t have all the antibodies for all the proteins

Almost omic for yeasts

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

What type of approach is used in mass sectromertry ?

A

Non-prescriptive

Identifies all the detectable proteins in a sample

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

What are mass spectrometry results based on?

A

Peptide m/z values

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

Are mass spectrometers classified as omic technologies?

A

Yes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
34
Q

What are the two proteomic technologies used?

A

Protein arrays

Mass spectrometry

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
35
Q

What is proteomic characterisation?

A

The characterisation and quantification of all polypeptide components present in a biological compartment

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
36
Q

What are the three levels by which proteins are characterised?

A

Primary

Secondary and tertiary

Quarternary

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
37
Q

What is the primary structure of proteins?

A

Amino acid sequence

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
38
Q

What is the quarternary structure of proteins?

A

Protein-protein interactions

Interactome

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
39
Q

What are the questioned addressed by proteomics?

A

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?

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
40
Q

What are the pros of mRNA profiling?

A

Fairly omic

Quick

Relatively cheap

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
41
Q

What are the pros of proteomics?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
42
Q

What are the cons of mRNA profiling?

A

Bad at looking at certain ECM and fluids

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
43
Q

What are the cons of proteomics?

A

Difficult to perform

Expensive

Not truly comprehensive

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
44
Q

Do proteomes and mRNA profiles always correlate?

A

Some correlate well, some don’t

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
45
Q

What are the two reasons for why we need sample fractionation?

A

Dynamic range

Complexity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
46
Q

Why does the dynamic range of a sample mean we need sample fractionation?

A

Some molecules very abundant in the serum

This is too large for a single detector

47
Q

Why does the complexity of a sample mean we need sample fractionation?

A

A large number of protein sequences

1 000 000 protein sequences

This is too large for a single platform

48
Q

What does proteomic mass spectrometry analyse?

A

Peptides produced by proteolytic digestion of samples

49
Q

What important step is crucial to perform before fractionation?

A

Targeted depletion of abundant proteins

So they don’t interfere with results

Immunoaffinity and ligand binding

50
Q

What are the two types of non-targeted fractionation?

A

Protein fractionation

Peptide fractionation

51
Q

Two types of protein fractionation

A

1D or 2D electrophoresis

1D or 2D-liquid chromatography

52
Q

Two types of peptide fractionation

A

Solution phase isoelectric focusing

2D HPLC

53
Q

What is the key in fractionation?

A

Minimise experimental error

54
Q

What are two ways to minimise the experimental error in fractionation?

A

High reproducibility

Avoid non-specific losses

55
Q

What are the two types of variation that exist in experiments?

A

Technical variation

Biological variation

56
Q

What is technical variation?

A

Experimental error

Occur whenever we prepare or analyze samples

57
Q

What are ways to reduce technical variation?

A

Standard operating procedures

Quantifying using technical replicates

58
Q

What are sources of biological variation?

A

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
Q

What are ways to reduce biological variation?

A

Increase cohort size

Stratification - cluster variation

Quantify using biological variations

60
Q

Biological samples are often impure

TRUE or FALSE

A

TRUE

Due to contamination and cell and tissue heterogeneity

61
Q

What are the reasons we need validation?

A

Variation (biological and technical)

Proteomic results are based on probability

Important identifications require validations using orthogonal techniques

62
Q

What does validation due?

A

Make sure the identity of the sample is correct and it localises to the tissue/cell/organelle

63
Q

What are orthogonal techniques?

A

Completely different technique than the one used in the experiment

64
Q

What are examples of orthogonal techniques?

A

Immunohistochemistry

ELISA

Labelling

65
Q

Why are mass spectometers used over ELISAS and protein blocks?

A

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
Q

What are the steps of mass spectometers?

A

Preparation

Mass-spectometer

Protein identification

Analysis/bioinformatics

Validation

67
Q

Describe the preparation steps taken before the sample is inserted into the mass spectometer

A

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
Q

Give an example of a validation technique

A

Identify a biomarker for ovarian cancer

Look at serum of patients and compare it to healthy controls

Then validate these using orthognal techniques

69
Q

Why do we need to digest the proteins?

A

In order to be able to fragment and ionise them

70
Q

Can we identify a protein using its ionised form?

A

No

71
Q

Can we identify a protein using its fragmented form?

A

No

72
Q

Can we identify a protein using the ionised form of its peptide?

A

No

73
Q

Can we identify a protein using the fragmented form of its peptide?

A

Yes

74
Q

What is the challenge with ionising proteins?

A

Very high energy needed for direct desorption and ionisation

75
Q

What is the challenge with fragmenting proteins?

A

Difficult to produced ordered series of daughter ions producing any amino acid sequence information

76
Q

What is required in order to ionise peptides?

A

High energy

77
Q

What is the usefulness of the fragmented peptides?

A

Peptide fragmentation data can be interpreted to deduce amino acid sequence information

78
Q

What are we measuring in a mass spectrometer?

A

The mass to charge ratio of peptides fragmented from the proteins we want to analyse

79
Q

Describe how liquid chromatography works in collaboration with mass spectometry

A

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
Q

What does the mass filter do?

A

Guides the ions straight into the mass spec

81
Q

What is the purpose of the separation techniques in mass specs?

A

Prevents the molecules from clustering together

Separates them by size or chemical properties

82
Q

Why do we break proteins down into peptides in mass spec?

A

They are more stable

Can identify the peptides, which is used to quantify and identify whole proteins

83
Q

What charge do proteins have following soft ionisation?

A

Positive charge

84
Q

What is the benefit of the positive charge of proteins?

A

Helps filter out other substances

85
Q

How do mass specs measure the mass to charge ratio of peptides?

A

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
Q

Singularly charged peptides moves in vacuums with half the force as peptides with double charges

TRUE or FALSE

A

TRUE

87
Q

What are two techniques of soft ionisation?

A

Matrix-assisted laser desorption ionisation

Electrospray ionisation with water

88
Q

Describe the process behind MALDI

A

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
Q

What does the time of flight in MALDI represent?

A

Amount of time taken for the ionised molecule to hit detector on the back

90
Q

What are the two techniques used in ESI?

A

Quadrupole

Orbitrap

91
Q

Describe the components of the quadrupole

A

Contains 4 poles

2 positive and 2 negative

92
Q

How does a quadrupole work in identifying peptides?

A

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
Q

Describe the structure of the orbitrap

A

Outer barrel-like electrode

Coaxial inner spindle-like electrode that traps ions in an orbital motion around the spindle

94
Q

How does the orbitrap work?

A

Ions are trapped because their electrostatic attraction to the inner electrode is balance by their inertia

95
Q

What are the two types of mass spectra obtained following fragmentation?

A

Single stage MS

Tandem MS

96
Q

What information is obtained from single stage MS?

A

Protein mass data, including purity and integrity

Peptide mass data using the fingerprint

97
Q

What is the use of single stage MS?

A

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
Q

What is the use of tandem MS?

A

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
Q

What charge do tryptic peptides possess normally?

A

Double

100
Q

What types of ions are produced from the ionisation of tryptic peptides?

A

Single charged

b- and y- ions

101
Q

Where are the b-ions retained?

A

In the N-terminal fragment

102
Q

Where are the y-ions retained?

A

In the C-terminal fragment

103
Q

What are 3 ways of quantifying peptides?

A

SILAC

Label free

Chemical mass tagging

104
Q

What is SILAC?

A

Stable isotope labelling of amino acids in culture

Gold standard

105
Q

How is SILAC carried out?

A

Grow cells in heavy medium

106
Q

What is the disadvantage with SILAC?

A

Does’t work in animal or human experiments

Too fast or not possible

107
Q

Examples of label free quantification mechanisms

A

iBAQ

Top3

EmPAI

108
Q

What is the advantage of label free quantification mechanisms?

A

Does not modify proteins, so no risk of affecting ionisation or properites

109
Q

What is the disadvantage of label free quantification mechanisms?

A

Have to do one sample at a time

110
Q

What are examples of chemical mass tagging used to quantify proteins?

A

TMT

iTRAQ

111
Q

Describe the mechanism behind chemical mass tagging

A

Process of chemically modifying peptides according to their mass

112
Q

What is the advantage of chemical mass tagging?

A

You can have different weighted tags which makes it high throughput

113
Q

What is the most ideal quantification technique?

A

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.
© 2024 Bold Learning Solutions. Terms and Conditions