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SCII > Bioinformatics > Flashcards

Bioinformatics Flashcards

1
Q

What is a proteoform?

A

A proteoform is a specific form of a protein that includes its post-translational modifications, alternative splice variants, and any other sources of combinatorial variation.

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2
Q

What does proteomics study?

A

The large-scale study of proteins, their structures, functions, modifications and interactions

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3
Q

Which technique is primarily used for identifying proteins in proteomics?

A

Mass spectometry

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4
Q

How does mass spectometry separate molecules?

A

Based on their mass-to-charge ration (m/z)

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5
Q

Wat is the principle of Guilt-by-Association in proteomics?

A

The idea that if a protein consistently interacts with another protein, they likely share a related function

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6
Q

Why is plasma proteomics challenging?

A

Due to the dynamic range of protein concentrations, from highly abundant proteins like albumin to very low-abundance proteins like interleukins

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7
Q

What is the general workflow of proteomics?

A
  1. Protein isolation
  2. Digestion into peptides
  3. Peptide separation (via liquid chromatography)
  4. Fragmentation
  5. Mass spectometry analysis
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8
Q

How is proteomics applied in clinical research?

A

!. Study tumor tissues
2. Identify biomarkers in diseases
3. Plasma profiling for detecting disease-related proteins

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9
Q

What are HLA peptides, and why are they importantin immunoproteomics?

A

HLA peptides are fragments of proteins presented by HLA molecules, crucial for immune recognition and developing immunotherapies

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10
Q

Why is proteomics considered a better alternative to western blotting?

A

More sensitive, provides quantification, doesn’t rely on antibodies, which can sometimes be unreliable or non-specific in western blotting

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11
Q

What is targeted proteomics, and why is it used?

A

Used to focus on specific peptides for specific quantification. It is more acurate and sensitive compared to traditional methods. One example is parallel reaction monitoring.

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12
Q

What role do post-translational modifications play in proteomics?

A

Post-translational modifications such as phosphorylation and glycosylation alter protein functions and are a key focus in proteomics for understanding protein activity in different conditions.

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13
Q

What is the advantage of mass spectometry in detecting post-translational modifications?

A

It can accurately detect and identify post-translational modifications, which are often difficult to study using traditional methods like western blotting

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14
Q

What does the term “unbiased” mean in the context of proteomics?

A

Proteomics i considered unbiased because it can identify unknown proteins without pre-selecting specific targets, unlike conventional methods that focus only on known proteins.

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15
Q

How can proteomics be used to develop personalized therapies?

A

It can help identify tumor- or virus-specific HLA peptides, leading to the development of personalized immunotherapies targeting those peptides.

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16
Q

What are the four basic functions of a mass spectrometer?

A

Charge, accelerate, separate, and measure ions

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17
Q

What does MALDI stand for?

A

Matrix-Assisted Laser Desorption/Ionization

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18
Q

What does mass spectrometry do in simple terms?

A

It sorts neutral molecules into ions and then sorts them according to their masses

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19
Q

What is the principle behind ions with smaller mass in a mass spectrometer?

A

Smaller ions travel faster than larger ions when subjected to the same force

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20
Q

What does MALDI-TOF stand for?

A

Matrix-Assisted Laser Desorption/Ionization - Time of Flight

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21
Q

Why is rapid identification of bacteria important in clinical settings?

A

Knowing the bacteria type can help predict antimicrobial susceptibility even without testing, speeding up treatment decisions

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22
Q

How does mass spectrometry assist in determining antibiotic resistance?

A

It detects measurable shifts in the mass of bacterial components after interaction with antibiotics, indicating resistance

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23
Q

What is the significance of the beta-lactam ring in antibiotics?

A

It is the key structure in beta-lactam antibiotics that inhibits bacterial cell wall synthesis

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24
Q

How does the beta-lactamase enzyme contribute to antibiotic resistance?

A

Beta-latamase breaks down beta-lactam antibiotics by hydrolyzing the beta-lactam ring, rendering the antibiotic ineffective

25
Q

What are the advatnages of using mass spectrometry over PCR in detecting antibiotic resistance?

A

Mass spectrometry provides phenotypic results, showing actual resistance activity rather than just the presence of resistance genes, which may or may not be expressed

26
Q

What is the main advantage of high-resolution mass spectrometry like Orbitrap?

A

It can analyze ions with high sensitivity and resolution, making it useful for detecting specific bacterial peptides and proteins related to antibiotic resistance

27
Q

What is trypsin used for in mass spectrometry experiments?

A

Trypsin is an enzyme used to cleave proteins into smaller peptides for mass spectrometric analysis

28
Q

How does MALDI-TOF help in bacterial identification?

A

It produces a spectrum of ribosomal proteins that can be matched to a database to identify bacterial species

29
Q

What is immune repertoire sequencing?

A

The sequencing of the variable regions of the T cell receptor (TCR) and the B cell receptor (BCR). It helps analyze the diversity and specificity of immune cells.

30
Q

What are the three processes that change the immune repertoire?

A
  1. VDJ recombination (applies to both B cells and T cells)
  2. Somatic hypermutation (applies to B cells)
  3. Class switch recombination (applies to B cells)
31
Q

What is VDJ recombination?

A

VDJ recombination is the process by which variable (V), diversity (D), and joining (J) gene segments are rearranged to create a diverse repertoire of TCRs and BCRs.

32
Q

How is diversity generated in immune receptors?

A
  1. Combinational diversity (different V, D, and J gene combinations)
  2. Junctional diversity (nucleotide deletions, insertions, mediated by TDT enzyme)
  3. Combinations of heavy and light chains in BCRs.
33
Q

What is somatic hypermutation (SHM)?

A

SHM is the process by which point mutations are introduced in the variable region of the BCR genes, leading to increased affinity for the antigen. It occurs mainly in the CDR regions.

34
Q

What is the role of TDT in immune diversity?

A

It is an enzyme that adds random nucleotides to the junctions between V, D, and J gene segments during recombination, contributing to junctional diversity.

35
Q

What is class switch recombination (CSR)?

A

CSR is the process in which a B cell changes the class of antibody it produces (eg. from IgM to IgG or IgA) by recombining the constant region of the immunoglobulin heavy chain gene.

36
Q

What are CDR regions in immune receptors?

A

Complementarity-detemining regions are parts of the variable regions of BCRs and TCRs that interact with the antigen, determining specificity. Mutations in these regions can increase antigen affinity.

37
Q

What is the difference between analyzing BCR and TCR repertoires at the DNA vs. RNA level?

A

At the DNA level, both functional and non-functional rearrangements are captured, while ast the RNA level, only expressed functional rearrangements and the constant region can be analyzed.

38
Q

What is IMGT, and why is it important for immune repertoire analysis?

A

IMGT is a global database of immunoglobulin and TCR gene sequences, serving as the reference for analyzing immune repertoire data. It provides up-to-date information on V, D, and J genes.

39
Q

Which process introduces nucleotide additions and deletions during receptor formation, and does it apply to B cells, T cells, or both?

A

VDJ recombination in TCRs and BCRs.

40
Q

Which proces involves mutations in the variable region of the receptor to increase antigen binding affinity, and does it apply to B cells, T cells, or both?

A

Somatic hypermutation (SHM) in B cells.

41
Q

What are three considerations when determining which sequencing technique to apply in immune repertoire analysis?

A

DNA vs. RNA: DNA captures functional and non-functional rearrangements; RNA focuses on expressed functional genes and allows constant region analysis.
Number of cells: Large-scale repertoire sequencing can analyze thousands to millions of rearrangements, while single-cell sequencing is more limited byt provides detailed insights.
Research focus: Whether you need to focus on bulk repertoire or single-cell analysis will impact the choice of sequencing platform.

42
Q

What is one consideration when deciding whether to sequence at the DNA or RNA level for immune repertoire analysis?

A

If you want to study both functional and non-functional rearrangements, sequencing at the DNA level is preferred. If you want to study constant regions and functional transcripts, RNA sequencing is more suitable.

43
Q

What sequencing technique should be applied if you want to analyze the repertoire of a large population of cells for broad diversity?

A

Parallel next-generation sequencing (NGS), as it can handle thousands to millions of rearrangements efficiently

44
Q

What sequencing technique should be applied if the goal is to study both heavy and light chains from individual B or T cells?

A

Single-cell sequencing, as it allows for detailed pairing of receptors in a single cell.

45
Q

What sequencing technique would be best for identifying immune repertoire changes in a small subset of cells with known rearrangements, such as leukemia?

A

Single-cell sequencing, as it provides precise information on the clonotypes and rearrangements of individual cells

46
Q

Why is NGS a preferred method for large-scale immune repertoire analysis?

A

It is high-throughput, relatively cost-effective, and capable of sequencing a vast number of unique rearrangements, providing a comprehensive view of immune diversity.

47
Q

What are the strengths of amplicon-based NGS for immune repertoire sequencing? (3)

A

It allows for (1) deep sequencing of specific immune receptor regions, (2) provides high accuracy in detecting clonality, and (3) gives a detailed quantification of B and T cell receptors.

48
Q

What are the weaknesses of amplicon-based NGS?

A

It is limited to specific regions (BCR or TCR), which may miss broader mutations or rearrangements in other parts of the genome. It also cannot capture full immune receptor diversity across all chains

49
Q

What are the strengths of capture-based NGS for immune repertoire sequencing?

A

It provides a broader view by allowing the sequencing of multiple regions of interest, including immune receptor genes and oncogenes, and is useful for detecting somatic hypermutations, copy number variations, and oncogenic mutations.

50
Q

What are the weaknesses of capture-based NGS?

A

It offers lower sequencing depth compared to amplicon-based NGS, making it less effective for highly detailed clonality analysis. It also requires more complex preparation, including DNA fragmentation and hybridization steps.

51
Q

What are the strengths of single-cell sequencing in immune repertoire analysis?

A

It allows for pairing of heavy and light chains (B cells) or alpha and beta chains (T cells) within individual cells, provides high-resolution data, and links immune receptor data to specific cell types.

52
Q

What are the weaknesses of single-cell sequencing?

A

It is lower throughput, more expensive, and time-consuming compared to bulk sequencing techniques. It also requires cell sorting and specialized equipment for isolating single cells.

53
Q

When would you apply amplicon-based NGS?

A

It is ideal for detailed clonality analysis, particularly when you need to focus on specific immune receptor regions and identify the frequency of particular clonotypes, such as in leukemia or lymphoma.

54
Q

When would you apply capture-based NGS?

A

When you need to analyze multiple regions of the genome, such as immune receptor genes and oncogenes, or when you’re looking for broad mutational profiles such as in cancer genomics.

55
Q

When would you apply single-cell sequencing?

A

When you need to determine paired heavy and light chains or alpha and beta chains from the same cell, or when studying specific immune cell populations in diseases like autoimmunity or lymphoproliferative disorders.

56
Q

How do aplicon-based and capture-based NGS compare?

A

Amplicon: deep sequencing of specific regions
Capture: broader overview of immune receptor genes and oncogenes, but with less depth

Amplicon detailed clonality
Capture: Broad mutational landscapes

57
Q

What benefits can be achieved by combining amplicon-based and capture-based NGS?

A

Allows for deep analysis of specific clonotypes while simulateously identifying broad mutational profiles and structural variations, providing a comprehensive view of immune responses and mutations

58
Q

What factors should be considered when choosing an immune repertoire sequencing technique? (4)

A
  1. Sample availability: enough DNA, RNA or sorted cells?
  2. Research question: is the focus on clonality, mutations or cell-type specific responses?
  3. Budget
  4. Material type: DNA or RNA? ex. RNA may be better for styding isotype switching in B cells.
59
Q

What should you consider if you have limited sample availability?

A

Amplicon-based NGS. It required less input and focuses on specific regions. If you want to capture broader information, you may opt for capture-based NGS, but you may need more sample material.

SCII (7 decks)

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