El Georgio

Question 1

What is mass spectrometry?

Answer 1

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

Question 1

What is proteomics

Answer 1

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

Question 2

What is mass spectrometry-based proteomics

Answer 2

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

Question 3

What is a key mechanism of protein activation

Answer 3

Post-translational modifications

Question 4

T/F the proteome of the cell is static

Answer 4

False, dynamic

Question 5

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

Answer 5

Question 6

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

Answer 6

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

Question 7

Why is top down not suitable for most biomedical applications

Answer 7

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)

Question 8

Advantages and disadvantages of bottom-up proteomics

Answer 8

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

Question 9

What is the prototypical proteomics pipelien

Answer 9

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

Question 10

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

Answer 10

Question 11

Discovery phase workflow

Answer 11

Question 12

Validation phase workflow

Answer 12

Question 13

What is the most important step

Answer 13

Sample preparation

Question 14

Goals of sample preparation

Answer 14

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

Question 15

Which levels of proteins are well-studied

Answer 15

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.

Question 16

What is the multiple affinity removal system (MARS)

Answer 16

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

Question 17

What is immunodepletion

Answer 17

Depletes samples of abundant plasma proteins

Uses immobilized antibodies

Question 18

Which AAs do trypsin cut? What pH?

Answer 18

C-term to Lys, Arg
pH 8.5

Question 19

6 steps of In-Solution Tryptic Digestion

Answer 19

Question 20

How does a mass spectrometer work

Answer 20

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

Question 21

What is MALDI? What are the steps?

Answer 21

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

Question 22

Pros and cons of MALDI

Answer 22

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

Question 23

What is ESI

Answer 23

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

Question 24

Pros and Cons of ESI

Answer 24

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)

Question 25

What is time of flight

Answer 25

* Ions fly along a long tube * Diagram shows a reflector * Smaller ions get to the detector 1st * Analogous to a gel

Question 26

What is Quadrupole

Answer 26

* 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

Question 27

What is ion trap

Answer 27

* 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

Question 28

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

Answer 28

Threshold dissociation (Weakest bonds are broken) Collision induced dissociation: Weakest Infrared multiphoton dissociation: Weakest Electron capture dissociation: Random Electron transfer dissociation: Random

Question 29

What is collision induced dissociation?

Answer 29

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

Question 30

What is infrared multiphoton dissociation?

Answer 30

Use infrared laser to break bonds

Question 31

What is electron capture dissociation?

Answer 31

Multiple charged cations capture electrons, inducing odd-electron fragmentation

Question 32

What is electron transfer dissociation?

Answer 32

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.

Question 33

What is monoisotopic mass

Answer 33

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

Question 34

Why use trypsin for MS-MS

Answer 34

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

Question 35

How do peptides break apart under low energy dissociation conditions

Answer 35

Fragment at the C - N bond

Question 36

b vs y- ion

Answer 36

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

Question 37

what are a-type ions

Answer 37

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

Question 38

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

Answer 38

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+)

Question 39

Things to keep in mind about tryptic peptides

Answer 39

y1 will either be lysine at 147 or arginine at 175

Question 40

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

Answer 40

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

Question 41

Steps of spectrum mill

Answer 41

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

Question 42

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

Answer 42

Question 43

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

Answer 43

Well suited to answer biologic questions Used as a tool to evaluate protein distributions

Question 44

Pros and cons of SILAC

Answer 44

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

Question 45

What is iTRAQ

Answer 45

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