Mass Spectrometry Flashcards
What is mass spectrometry
What are the three basic parts of a mass spectrometer
State the different ionisation methods that can be used for mass spec
Explain electron impact ionisation
Write the general equation for ionisation
Explain Matrix Assisted Laser Desorption Ionisation (MALDI)
What lasers can be used for MALDI
What are the common MALDI matrices
How do modern MALDI instruments differ from early ones
What problem does delayed extraction solve
How does Delayed Extraction (DE) ensure ions have the same velocity when they enter the analyser
What is electro spray ionisation (ES or ESI)
How does ESI work
What is nano-electrospray
What different types of mass analysers are there + 3 their key factors
What are quadrupole mass filters
How do quadrupole mass filters work
What are the characteristics of quadrupole mass filters
What are ion trap analysers
How do ion trap analysers work
What are orbitrap mass analysers
What are the characteristics of orbitraps
What are TOF analysers
What is a reflectron
What are the different types of ion detectors in a mass spectrometer
Detects ions and provides quantitative info, tells you how much of an ion species is in the mixture
It amplifies the signal you get which increases the sensitivity
PM-photomultiplier: detects photons
EM-electron multiplier
MCP- micro-channel plate array
detectors
What do PM detectors do
What do EM detectors do*
How do MCP ion detectors work
What are the more commonly used components of a mass spectrometer
MALDI sources are most commonly found on instruments with TOF
analysers
• MALDI-TOF is the method of choice for “mass fingerprinting” complex
mixtures of polymers – both chemical and biological
ES sources are most commonly found on triple quadrupole, ion trap, orbitrap
or Q-TOF instruments (more on this later)
• ES is especially useful for multiply charged molecules eg peptides and
proteins
What are hybrid MS instruments
What are the different types of molecular ions *
What are fragment ions
What are alkaloids
What is collisional activation
Ionise sample
ions are then accelerated into the collision cell or collision chamber under a high voltage.
Collision with Gas Molecules:
The accelerated ions enter the collision chamber, where they collide with inert gas molecules (e.g., argon or nitrogen) at high velocity. This collision transfers energy to the ions.
What is MS/MS
Explain how a triple quadrupole works
How does a Q-TOF MS work*
How does a MALDI TOF- TOF MS work
The matrix absorbs the energy from a laser and transfers that energy to the sample, causing the sample molecules to desorb (get released) and ionize. The ionization process results in charged particles (ions) of the sample.
a TOF analyzer, ions are accelerated by an electric field and then travel through a flight tube to a detector.
The flight time of each ion is proportional to its mass-to-charge ratio (m/z), with heavier ions taking longer to reach the detector.
The second TOF analyzer comes into play after the initial TOF spectrum is generated. In the TOF-TOF setup, ions are subjected to collision-induced dissociation (CID) or a similar fragmentation technique in a collision cell between the two TOF analyzers. Here, ions are collided with neutral gas molecules (e.g., nitrogen or argon), which causes the ions to fragment into smaller pieces.
The fragments are then accelerated into the second TOF analyzer, where they are detected based on their new flight times, yielding another mass spectrum.
How does ES-MS/MS Peptide Sequencing work
How can you recognise different multiply charged ions*
Divide m/z value of molecular ion by amount of protons added to produce the fragment ion
How are peptides fragmented for sequencing
Can sequence a peptide using mass spec because peptides aren’t symmetrical molecules
They have different things on different ends ( N and C terminus)
Add a proton to generate singly charged molecular ion to where the bond will be cleaved
What is an a-ion
What is a y-ion
Predicted b and y’’ fragment ions: not all will be present in the spectrum.
Fragmentation of singly charged molecular ions in MALDI-MS/MS
experiments similarly results in b and y” fragmentation but the ion
abundances are often very different from ES-MS/MS.
y-ions: Contain the C-terminal part of the peptide (including the terminal -COOH).
What is determined in proteomics experiments
What is 2D PAGE
Why is gel electrophoresis used
What is used for detection in 2D PAGE
Explain the proteomics fingerprinting strategy
Explain the basic proteomics sequencing pathway
What is shotgun proteomics + disadvantages of 2D PAGE
What is MudPIT
•Applied to yeast Saccharomyces cerevisiae
•2D-PAGE identified 279 proteins
•MudPIT identified 1,484 proteins
•Including proteins of pI 3.82 and 12.55,
MW 559 kDa and membrane associated
Proteins
“Micro-MudPIT” 4984 proteins
were detected, 2/3 predicated
reading frames of yeast genome
What are PTMs
PTMs are the chemical modification of a protein after its translation.
They have profound effects on protein function by altering their activity state, localization, turnover, and interactions with other proteins.
Over 200 different types of PTM, every amino acid can be modified.
The majority of proteins are modified?
What are mod forms of proteins
What is one role of sugars
What is the glycocalyx
What are the different monosaccharides
How are glycosidic bonds formed
What are glycoproteins
What are the two types of protein glycosylation
What are the characteristics of protein N glycosylation
What is the general structure of N glycans
All N-glycans share a conserved core structure: two N-acetylglucosamine (GlcNAc) and three mannose (Man) sugars forming the core pentasaccharide linked to the nitrogen of an Asn residue.
Initiation (ER - Endoplasmic Reticulum)
Starts with synthesis of a lipid-linked oligosaccharide (Dolichol-P-P-oligosaccharide).
A 14-sugar oligosaccharide is built:
(Glc)₃(Man)₉(GlcNAc)₂ on dolichol phosphate.
This whole structure is transferred en bloc to an Asn residue on a nascent protein.
Glucosidases remove the 3 glucose residues.
Some mannoses may also be trimmed off.
core structure is modified into 3 types of N-glycans:
Common N-Glycan Extensions:
GlcNAc: Adds branches.
Galactose (Gal): Extends branches.
Fucose (Fuc): Often added to the core (core fucosylation).
Sialic acid (Neu5Ac): Caps the branches; important for charge & recognition.
Explain the N-glycan biosynthesis pathway
What are the different types of N glycans
High-mannose: Only mannose residues added.
Complex: Mannoses trimmed and replaced by other sugars (e.g., GlcNAc, galactose, sialic acid, fucose).
Hybrid: One arm looks like high-mannose; the other like complex-type.
What is commonly found in the antenna of N-glycans
What are N-glycan antenna capped with + how is blood groups determined
What is a main characteristic of glycans
What is O-glycosylation
What is the structure of O-glycans
O-glycans don’t have a common core shared by all types
Core 1 Galβ1–3GalNAcα1–O–Ser/Thr
Core 2 GlcNAcβ1–6(Galβ1–3)GalNAcα1–O–Ser/Thr
Core 3 GlcNAcβ1–3GalNAcα1–O–Ser/Thr
Core 4 GlcNAcβ1–6(GlcNAcβ1–3)GalNAcα1–O–Ser/Thr
What are mucins
What is the characteristics of O-GlcNac proteins
What are the Factors affecting glycosylation
Why are proteins glycosylated
What are glycoforms
What does sperm-egg recognition involved
What are glycodelins
Roles in reproductive system
4 isoforms
What is the roles of glycodelin- A
What are the roles of glycodelin-S
What is the differences between glycodelin A and S
Glycodelin-A:
Highly sialylated and fucosylated complex-type N-glycans
These sugars help it interact with immune receptors like DC-SIGN, contributing to immunosuppression.
Prevents maternal immune cells from attacking the fetus.
Glycodelin-S:
Simpler glycans, less sialylation
These modifications allow it to interact with sperm, aiding in capacitation (a process that makes sperm capable of fertilizing an egg).
Does not suppress immune activity.
What are the characteristics of the influenza virus
How does influenza infect cells
What do haemagglutin receptors bind to
What human influenza pandemics were there
How did Human Pandemic H1N1 arise
20-27% of the population infected, death rate of <0.02%
Give some examples of neuraminidase inhibitors
Which amino acids are highly conserved in influenzas NA’s
How does Tamiflu work
It binds to the active site of the neuraminidase enzyme.
It cleaves sialic acid residues on host cells and viral particles.
What is glycomics
Explain the glycomics screening strategy
What is added to glycans during glycomics
Which three monosaccharides would have the same m/z ratio
What can affect glycosylation
How does N-glycan MS/MS work *
What is Glycoproteomics used for
What’s the Glycoproteomics pathway
