Chapter 9 - Fundamentals of proteomics

Question 1

Amino acids contain both ________ and _________ functional groups.

Answer 1

amine, carboxyl

Question 2

How many standard alpha-amino acids are there?

Answer 2

20

Question 3

A series of alpha-amino acids joined by peptide bonds forms a

Answer 3

polypeptide chain.

Question 4

Each amino acid unit in a polypeptide is called a

Answer 4

residue.

Question 5

An oligopeptide has between __________ amino acids.

Answer 5

30-50

Question 6

A protein is typically more than _____ amino acids long.

Answer 6

50

Question 7

The convention for writing peptide sequences is to put the ______ on the left.

Answer 7

amino-terminus

Question 8

Basic amino acids

Answer 8

arginine, histidine, lysine

Question 9

Amino acids that can be phosphorylated

Answer 9

serine, threonine, tyrosine

Question 10

Amino acid to form O-linked glycosylation

Answer 10

serine, threonine

Question 11

Amino acid to form N-linked glycosylation

Answer 11

asparagine

Question 12

Amino acid containing a sulfhydryl group

Answer 12

cysteine

Question 13

The primary structure of a protein is

Answer 13

the sequence of amino acids that constitutes its structure.

Question 14

The secondary structure of a protein is the

Answer 14

three-dimensional shape adopted by the protein due to intra-molecular interactions.

Question 15

Typical examples of the secondary structure of a protein.

Answer 15

The formation of loops or helices

Question 16

The tertiary structure of a protein describes

Answer 16

all aspects of the three-dimensional folding of a polypeptide.

Question 17

Quaternary structure

Answer 17

The relative arrangement in space when a protein has two or more polypeptide subunits.

Question 18

Mass analysis provides a means of getting information relevant to the _______ structure of a protein.

Answer 18

primary

Question 19

One of the main challenges to overcome in MS proteomics research is

Answer 19

discovering the correct order of the amino acids that constitute the protein or peptide.

Question 20

Two proteins or polypeptides that are composed of the same amino acids but in different order will have

Answer 20

completely different properties.

Question 21

In MALDI, a solution of sample/matrix is placed on

Answer 21

a target plate and subsequently crystallized.

Question 22

In MALDI, the plate is positioned in

Answer 22

the high vacuum source region.

Question 23

In MALDI, the plate is irradiated with a

Answer 23

pulsed laser beam.

Question 24

In MALDI, the laser causes localized ______ of the matrix crystals.

Answer 24

sublimation

Question 25

In MALDI, the matrix molecules are ionized after

Answer 25

it absorbs energy from the laser beam.

Question 26

In MALDI, analyte molecules are ionized as

Answer 26

proton transfers from the matrix ions to the analytes.

Question 27

MALDI is a _______ ionization.

Answer 27

soft

Question 28

In MALDI, ______ ions dominate the mass spectrum.

Answer 28

[M+H]⁺

Question 29

In MALDI, _______ lasers are the most common due to their ease of operation and low price.

Answer 29

UV

Question 30

In MALDI, _______ lasers are considered the standard.

Answer 30

N₂

Question 31

In MALDI, IR lasers like ______ or ______ are also used.

Answer 31

Er:YAG, CO₂

Question 32

In MALDI, the laser spot diameter at the surface of the sample varies from

Answer 32

5 to 200 micrometers.

Question 33

In MALDI, IR lasers have _______ fragmentation and _______ sensitivity than UV lasers.

Answer 33

less, lower

Question 34

Role of the solid matrix in MALDI

Answer 34

• Separate analyte molecules
• Minimize the sample damage from the laser and increase the efficiency of energy transfer from laser to analyte
• Makes the selection of laser wavelength irrelevant
• Enables intact gas-phase ions from non-volatile, thermally labile large molecules

Question 35

Matrix selection in MALDI

Answer 35

Requirements include strong absorbance at the laser wavelength, low enough mass to be sublimable, vacuum stability, ability to promote analyte ionization, solubility in solvents compatible with analyte, and lack of chemical reactivity.

Question 36

Good matrix for peptides

Answer 36

CHCA (alpha-cyano-4-hydroxy cinnamic acid)

Question 37

Good matrix for peptides and proteins

Answer 37

DHB (2,5-dihydroxy benzoic acid)

Question 38

Good matrix for proteins

Answer 38

sinapinic acid (3, 5-dimethoxy-4-hydroxycinnamic acid)

Question 39

In MALDI, _______ microliter saturated matrix solution is mixed with _______ microliter of analyte solution.

Answer 39

5-10

1-2

Question 40

MALDI is relatively _________ to contamination by salts, buffers, detergents, and so on compared to other ionization techniques.

Answer 40

less sensitive

Question 41

MALDI disadvantages

Answer 41

• Low shot-to-shot reproducibility
• Strong dependence on the homogeneity of the sample preparation
• Usually not used in quantitative analysis

Question 42

Pulsed ion sampling in MALDI is compatible with

Answer 42

Ion trap, orbitraps, and TOF.

Question 43

__________ is one of the most widely used instruments in proteomics.

Answer 43

MALDI/TOFMS

Question 44

In delayed pulsed extraction, the energy spread of the same m/z ions by MALDI is of

Answer 44

significant magnitude.

Question 45

As the mass of a peptide or protein increases, so does the probability for the presence of

Answer 45

¹³C

Question 46

Large molecules examined under ESI-MS conditions typically show _______ fragmentation.

Answer 46

little

Question 47

Large molecules examined under ESI-MS conditions usually contain a distribution of ________ charged ions.

Answer 47

multiply

Question 48

Protein sequencing begins with

Answer 48

its digestion using a pretease (proteolysis).

Question 49

Trypsin cleaves polypeptide chains on the ______ side of ______ and ______ residues unless they are linked to ______.

Answer 49

carboxyl, arginine, lysine, proline

Question 50

A protein that contains 9 lysine and 7 arginine residues will usually yield ________ peptides on digestion with trypsin.

Answer 50

16

Question 51

Tryptic peptides will end with either

Answer 51

arginine or lysine.

Question 52

With ESI/MS, tryptic peptides usually become _______ charged.

Answer 52

doubly

Question 53

In doubly charged tryptic peptides, one charge is from _________ and the other charge is from ________.

Answer 53

amino-terminus on the left

carboxyl terminus on the right due to R or K

Question 54

Tryptic peptides can have higher charge states if

Answer 54

there are missed cleavages or the presence of another basic amino acid like histidine.

Question 55

Under ESI+ conditions, protonation of peptides occurs at basic residues which are divided into the more basic

Answer 55

arginine, histidine, and lysine sites, and the less basic NH₂ terminus.

Question 56

Protons associated with the more basic sites tend to remain fixed and the charge is

Answer 56

localized.

Question 57

A proton associated with the NH₂ terminus may migrate by

Answer 57

internal solvation to any of the amide linkages.

Question 58

A mass spectrum of the resulting digest of products produces a

Answer 58

peptide fingerprint.

Question 59

An improvement of LC-ESI/MS/MS analysis of tryptic peptides can be obtained by performing LC-ESI/MS/MS with

Answer 59

data dependent acquisition (DDA).

Question 60

In DDA, as full scan mass spectra are acquired continuously in LC-MS mode, any ion detected as multiply charged and with a signal intensity above a pre-defined threshold will

Answer 60

trigger the mass spectrometer to switch over to MS/MS mode.

Question 61

In DDA, the mass spectrometer switches back and forth between

Answer 61

MS and MS/MS modes in a single LC run.

Question 62

Peptide molecules are fragmented by

Answer 62

collision induced dissociation (CID)

Question 63

Collision induced dissociation

Answer 63

collisions with neutral background gas molecules (nitrogen, argon, etc.)

Question 64

Low-energy collisions (ion trap, QQQ, QTOF) promote fragmentation of a peptide primarily along the __________.

Answer 64

peptide backbone

Question 65

At low energies, get mostly _________ ions of tryptic peptides.

Answer 65

b- and y-

Question 66

Peptide fragmentation which maintains the charge on the C terminus is designated as a

Answer 66

y-ion (+1 or +2 charge).

Question 67

Fragmentation which maintains the charge on the N terminus is designated as a

Answer 67

b-ion (+1 charge).

Question 68

MudPIT

Answer 68

multidimensional protein identification technology