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BMIS 310 > El Georgio > Flashcards

El Georgio Flashcards

1
Q

What is mass spectrometry?

A

The study of systems by the formation of gaseous ions, with or without fragmentation, which are then characterized by their mass-to-charge ratios and relative abundances

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1
Q

What is proteomics

A

The study of proteins. Identification, quantification, localization, modifications, interactions, activities, and ultimately their function

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2
Q

What is mass spectrometry-based proteomics

A

A highly powerful tool for protein identification and quantification. Complementary to other technologies and analysis methods.
Elucidate the structure and chemical properties of molecules

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3
Q

What is a key mechanism of protein activation

A

Post-translational modifications

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4
Q

T/F the proteome of the cell is static

A

False, dynamic

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5
Q

Overviews of top-down proteomics and bottom-up proteomics also mass limitations

A
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6
Q

What is top-down mainly used for? What is it useful for

A

Weight determination
Most useful for single proteins or relative simple mixtures.
Determines protein integrity and purity
Distinguish sequence variants
Determine extend and ID of various modifications

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7
Q

Why is top down not suitable for most biomedical applications

A

Requires highly specialized instrumentation
Data interpretation is far more difficult and less automated
Cannot be easily applied to complex biological samples
Breadth and depth of coverage of the proteome is orders of
magnitude less than for bottom-up proteomics
Problematic classes of proteins (membrane proteins; very large
proteins; disulfide rich proteins)
Other problems (aggregation; precipitation)

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8
Q

Advantages and disadvantages of bottom-up proteomics

A

Advantages:
Data acquisition easily automated
Fragmentation of tryptic peptides well understood
Reliable software available for analysis
Separation of peptides to create less complex subsets of the proteome for MS
analysis is far easier than for proteins (relates to breadth and depth of coverage)

Disadvantages:
Simple relationship between peptide and protein lost
Took highly complex mixture and made it 20-100x more complex
Puts high analytical demands on instrumentation

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9
Q

What is the prototypical proteomics pipelien

A
  1. Proteolytic digestion
  2. LC-ESI
  3. MS
  4. MS/MS
  5. Data analysis
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10
Q

Describe the level of biomarker candidates and statistical power in each of the following project milestones:
Protein identification, statistical analysis, target selection, target validation

A
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11
Q

Discovery phase workflow

A
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12
Q

Validation phase workflow

A
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13
Q

What is the most important step

A

Sample preparation

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14
Q

Goals of sample preparation

A

Concentrate dilute samples (speed-vac; lyophilization)
Remove salts / buffer exchange (filtration columns; dialysis membranes)
Remove detergents (spin columns - high sample losses; precipitation –
methanol, acetone)
Fractionate proteins: MW; Isoelectric Point
Isolate sub-proteomes: Depletion; Enrichment
Reduce, alkylate, digest
Ultimately – to reproducibly produce proteolytic peptides for LC/MS analysis

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15
Q

Which levels of proteins are well-studied

A

1-3 are well studied

Major goal is to look at the low abundance proteins, current technology allows us to look at the top three layers of proteins.

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16
Q

What is the multiple affinity removal system (MARS)

A

Individual antibody materials are mixed in selected percentages and packed into a column format to remove up to 14 high-abundant proteins

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17
Q

What is immunodepletion

A

Depletes samples of abundant plasma proteins

Uses immobilized antibodies

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18
Q

Which AAs do trypsin cut? What pH?

A

C-term to Lys, Arg
pH 8.5

19
Q

6 steps of In-Solution Tryptic Digestion

A
20
Q

How does a mass spectrometer work

A
  1. Creates ions from analyte molecules
  2. Separates ions by mass and charge (m/z)
  3. Detects the separated ions and determines their m/z
  4. Selects and fragments ions of interest to provide structural information (MS/MS)
  5. Collects the data in m/z spectrum
21
Q

What is MALDI? What are the steps?

A

Technique used to analyze large molecules

The analyte is dissolved in excess of matrix compound that absorbs efficiently at the laser wavelength. The matrix absorbs the laser and ionizes the sample. The laser energy strikes the analyte/matrix under vacuum and produces analyte and matrix ions

22
Q

Pros and cons of MALDI

A

Pros:
Soft ionization from condensed phase
Good for large (up to 500,000 Da) thermally labile compounds
Produces primarily singly charged ions
Low limit of detection
Tolerance of salts and buffers

Cons:
Not useful for analytes of low mass (matrix background <700 Da)
Analyte must be miscible with matrix
and solvent
Crystals are non-uniform leading to variations in ions observed and relative intensities
MS/MS difficult
Not easily compatible with LC

23
Q

What is ESI

A

The biomolecultes are fed into a metal capillary tube.

A high votlage is applied to the capillary.

Jet of highly charged droplets emerge from capillary tip
Solvent is evaporated letting multiple charged ions to enter the analyzer

24
Q

Pros and Cons of ESI

A

Pros:
Analysis of compounds with a MW up to about 200,000 Da is possible
ESI is very “sensitive”; typical sample amounts range from 250 fmol to 10 pmol
Multiple charging (compatible with MS/MS methods)
Low limit of detection
Easily adaptable to LC

Cons:
Not tolerant of salts and buffers, which reduce sensitivity
Ions observed and relative intensity depends on a variety of factors
Multiple charging (require interpretation and mathematical transformation)

25
Q

What is time of flight

A
  • Ions fly along a long tube
  • Diagram shows a reflector
  • Smaller ions get to the detector 1st
  • Analogous to a gel
26
Q

What is Quadrupole

A
  • Voltages on 4 rods are scanned as ions enter from source
  • For a certain voltage, only a narrow range of m/z will pass
  • Other m/z’s are rejected
  • Result: a Scanning Mass Filter
  • Usually used in a series of 3
27
Q

What is ion trap

A
  • Traps ions in a 2D field
  • Both MS and MS/MS analyses can occur
  • Ions are sequentially ejected to the detector
  • High sensitivity, Good range
28
Q

Common methods of fragmentation (which bonds are broken in each method)

A

Threshold dissociation (Weakest bonds are broken)

Collision induced dissociation: Weakest

Infrared multiphoton dissociation: Weakest

Electron capture dissociation: Random

Electron transfer dissociation: Random

29
Q

What is collision induced dissociation?

A

Apply an electric field to accelerate precursor ions and induce bond cleavage via collision with neutral gas molecules (He, Ar, N2)

30
Q

What is infrared multiphoton dissociation?

A

Use infrared laser to break bonds

31
Q

What is electron capture dissociation?

A

Multiple charged cations capture electrons, inducing odd-electron fragmentation

32
Q

What is electron transfer dissociation?

A

ETD induces fragmentation along the peptide backbone in a sequence-independent manner, leaving labile modifications linked to the peptide chain.

This greatly simplifies identification of modification sites. ETD produces primarily c- and z-type fragment ions that complement the b-and y-type ions produced by CID, increasing sequence coverage and protein IDs.
Achieve better analysis of proteins and their post-translational modifications.

33
Q

What is monoisotopic mass

A

The monoisotopic mass of a molecule is the sum of the accurate masses for the most abundant isotope of each element present

34
Q

Why use trypsin for MS-MS

A

CID of peptides less than 2-3 kD is most reliable for MS-MS studies – The frequency of tryptic cleavage guarantees that most peptides will be of this size

Trypsin cleaves on the C-terminal side of arginine and lysine. By putting the basic residues at the C-terminus, peptides fragment in a more predictable manner throughout the length of the peptide

35
Q

How do peptides break apart under low energy dissociation conditions

A

Fragment at the C - N bond

36
Q

b vs y- ion

A

If the charge is retained on the N-terminal end of the peptide, the ion is known as a b-type ion

If the charge is retained on the C-terminal end, the ion is termed a y-type ion

37
Q

what are a-type ions

A

The fragmentation energy in some instruments, especially triple quadrupoles or quadrupole-time-of-flight hybrids (Q-TOFs) is often sufficient to generate cleavage at the C-C bond as well, causing loss of CO from the b ion

38
Q

What will be the masses of the largest and smallest b-type and y-type ions

A

The largest y-type ion will appear anywhere between 57 to 186 amu below the mass of the precursor ion. (largest y ion = precursor ion – b1)
The smallest y ion will appear at the amino acid residue mass plus 19 amu. (y1 = one AA residue + the C-terminal OH + charge)
The largest b ion will be at 18 amu plus the residue mass below the precursor.
The smallest b ion will be at the residue mass + 1. (b1 = one AA + N-terminal H+)

39
Q

Things to keep in mind about tryptic peptides

A

y1 will either be lysine at 147 or arginine at 175

40
Q

How do you do a MS/MS database search? What’s the search engine?

A

Select peptides that equal the input mass
Theoretically fragment peptides
Compare theoretical fragments to acquired spectrum
Generate score
Rank by score and display best matches

Spectrum Mill

41
Q

Steps of spectrum mill

A

Data extraction
MS Data Search
MS Data Validation- Peptides
MS Data Validation- Proteins
Protein Summary Details- Parameters
Protein summary display

42
Q

What is the role of Q-TOF, Spectrum Mill, Mass Profiler Professional, Pathway Architect

A
43
Q

What is quantitative proteomics? What is it well suited for?

A

Well suited to answer biologic questions

Used as a tool to evaluate protein distributions

44
Q

Pros and cons of SILAC

A

Stable isotope labelling by amino acids in cell culture

Pros:
Deep, highly precise quantification
Works well in most cell lines
Works with all PTMs

Cons:
Limited plex level (3 max)
Cannot label humans!!!
Relatively Expensive

45
Q

What is iTRAQ

A

Isobaric tags for relative and absolute quantification

Allows us to compare the relative abundance of proteins from eight different samples in a single mass spec experiment

Uses up to 8 tag reagents that bind covalently to the N-terminus of the peptide and any Lysine side chains at the amine group (global tagging)
Each sample set is digested separately and then mixed with the specific iTRAQ tag

The iTRAQ labels are designed such that the same peptide from each sample has the same molecular weight and chromatographic retention time, but yields a different fragment or reporter ion when the MS/MS spectrum is acquired
The relative intensity of the reporter ion signals in an individual MS/MS spectrum is a direct measure of the relative levels of that peptide in each of the original samples

BMIS 310 (8 decks)

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