Chapter 9 - Fundamentals of proteomics Flashcards
Amino acids contain both ________ and _________ functional groups.
amine, carboxyl
How many standard alpha-amino acids are there?
20
A series of alpha-amino acids joined by peptide bonds forms a
polypeptide chain.
Each amino acid unit in a polypeptide is called a
residue.
An oligopeptide has between __________ amino acids.
30-50
A protein is typically more than _____ amino acids long.
50
The convention for writing peptide sequences is to put the ______ on the left.
amino-terminus
Basic amino acids
arginine, histidine, lysine
Amino acids that can be phosphorylated
serine, threonine, tyrosine
Amino acid to form O-linked glycosylation
serine, threonine
Amino acid to form N-linked glycosylation
asparagine
Amino acid containing a sulfhydryl group
cysteine
The primary structure of a protein is
the sequence of amino acids that constitutes its structure.
The secondary structure of a protein is the
three-dimensional shape adopted by the protein due to intra-molecular interactions.
Typical examples of the secondary structure of a protein.
The formation of loops or helices
The tertiary structure of a protein describes
all aspects of the three-dimensional folding of a polypeptide.
Quaternary structure
The relative arrangement in space when a protein has two or more polypeptide subunits.
Mass analysis provides a means of getting information relevant to the _______ structure of a protein.
primary
One of the main challenges to overcome in MS proteomics research is
discovering the correct order of the amino acids that constitute the protein or peptide.
Two proteins or polypeptides that are composed of the same amino acids but in different order will have
completely different properties.
In MALDI, a solution of sample/matrix is placed on
a target plate and subsequently crystallized.
In MALDI, the plate is positioned in
the high vacuum source region.
In MALDI, the plate is irradiated with a
pulsed laser beam.
In MALDI, the laser causes localized ______ of the matrix crystals.
sublimation
In MALDI, the matrix molecules are ionized after
it absorbs energy from the laser beam.
In MALDI, analyte molecules are ionized as
proton transfers from the matrix ions to the analytes.
MALDI is a _______ ionization.
soft
In MALDI, ______ ions dominate the mass spectrum.
[M+H]+
In MALDI, _______ lasers are the most common due to their ease of operation and low price.
UV
In MALDI, _______ lasers are considered the standard.
N2
In MALDI, IR lasers like ______ or ______ are also used.
Er:YAG, CO2
In MALDI, the laser spot diameter at the surface of the sample varies from
5 to 200 micrometers.
In MALDI, IR lasers have _______ fragmentation and _______ sensitivity than UV lasers.
less, lower
Role of the solid matrix in MALDI
- 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
Matrix selection in MALDI
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.
Good matrix for peptides
CHCA (alpha-cyano-4-hydroxy cinnamic acid)
Good matrix for peptides and proteins
DHB (2,5-dihydroxy benzoic acid)
Good matrix for proteins
sinapinic acid (3, 5-dimethoxy-4-hydroxycinnamic acid)
In MALDI, _______ microliter saturated matrix solution is mixed with _______ microliter of analyte solution.
5-10
1-2
MALDI is relatively _________ to contamination by salts, buffers, detergents, and so on compared to other ionization techniques.
less sensitive
MALDI disadvantages
- Low shot-to-shot reproducibility
- Strong dependence on the homogeneity of the sample preparation
- Usually not used in quantitative analysis
Pulsed ion sampling in MALDI is compatible with
Ion trap, orbitraps, and TOF.
__________ is one of the most widely used instruments in proteomics.
MALDI/TOFMS
In delayed pulsed extraction, the energy spread of the same m/z ions by MALDI is of
significant magnitude.
As the mass of a peptide or protein increases, so does the probability for the presence of
13C
Large molecules examined under ESI-MS conditions typically show _______ fragmentation.
little
Large molecules examined under ESI-MS conditions usually contain a distribution of ________ charged ions.
multiply
Protein sequencing begins with
its digestion using a pretease (proteolysis).
Trypsin cleaves polypeptide chains on the ______ side of ______ and ______ residues unless they are linked to ______.
carboxyl, arginine, lysine, proline
A protein that contains 9 lysine and 7 arginine residues will usually yield ________ peptides on digestion with trypsin.
16
Tryptic peptides will end with either
arginine or lysine.
With ESI/MS, tryptic peptides usually become _______ charged.
doubly
In doubly charged tryptic peptides, one charge is from _________ and the other charge is from ________.
amino-terminus on the left
carboxyl terminus on the right due to R or K
Tryptic peptides can have higher charge states if
there are missed cleavages or the presence of another basic amino acid like histidine.
Under ESI+ conditions, protonation of peptides occurs at basic residues which are divided into the more basic
arginine, histidine, and lysine sites, and the less basic NH2 terminus.
Protons associated with the more basic sites tend to remain fixed and the charge is
localized.
A proton associated with the NH2 terminus may migrate by
internal solvation to any of the amide linkages.
A mass spectrum of the resulting digest of products produces a
peptide fingerprint.
An improvement of LC-ESI/MS/MS analysis of tryptic peptides can be obtained by performing LC-ESI/MS/MS with
data dependent acquisition (DDA).
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
trigger the mass spectrometer to switch over to MS/MS mode.
In DDA, the mass spectrometer switches back and forth between
MS and MS/MS modes in a single LC run.
Peptide molecules are fragmented by
collision induced dissociation (CID)
Collision induced dissociation
collisions with neutral background gas molecules (nitrogen, argon, etc.)
Low-energy collisions (ion trap, QQQ, QTOF) promote fragmentation of a peptide primarily along the __________.
peptide backbone
At low energies, get mostly _________ ions of tryptic peptides.
b- and y-
Peptide fragmentation which maintains the charge on the C terminus is designated as a
y-ion (+1 or +2 charge).
Fragmentation which maintains the charge on the N terminus is designated as a
b-ion (+1 charge).
MudPIT
multidimensional protein identification technology
