Protein Science Notes (Mass spectrometry & Glycolysation)
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Course
Protein Science
Institution
Imperial College London (ICL)
Second year student studying for a Biochemistry/Biotechnology degree
Content:
- Protein dynamics by NMR
- Mass Spectrometry in great detail (includes ionisation sources, schematic diagrams, types of Mass spec like Quadrupole mass filters, etc.)
- Parts of a mass spectrometer (Mass analyser,...
Lecture Objectives of the Protein Science Module
Consolidate and extend your knowledge of
macromolecules (esp. proteins) and how to
investigate them
Beyond the textbook - link learning with research
Examples in lectures and practical class
Skills building
Structure/function relationships
Application in practical class
How to digest and present information (flash
presentations) Computer docking practical
Lecturers covering Protein Science
Prof. Stuart Haslam’s 5 Lectures
• Mass spectrometry
• Biomolecular structural elucidation
• Protein post-translational modification structure
and function
Konstantinos Beis’s 5 Lectures
• Lecture 1: Catalytic antibodies. New therapeutic
approaches to combat cocaine addiction
• Lecture 2: Introduction to antibody drug
conjugates
• Lecture 3-4: Principles of structure based drug
design
• Lecture 5: Drug metabolism. How does the human
body detoxifies drugs by enzymatic processes?
Ernesto Cota’s 5 Lectures
Protein-Ligand Interactions
• Introduction
• Forces involved
• Theoretical treatments
• Single binding site
• Multiple independent binding sites
• Data analysis
• How to measure protein-ligand interactions
• Library and analytical methods
• Practical considerations
• Comparison between techniques
Alfonso De Simone’s 5 Lectures
Protein Dynamics by NMR
The module will overview how NMR is used to
characterise protein dynamics. The lectures will range
from the general principles underlying protein NMR to
the characterisation of complex dynamics such as
those ocuring during enzymatic reactions.
,Protein Science 2 – Haslam Intro to Mass Spectrometry
A mass spectrometer is an instrument which is used
Outline of the Lectures on Mass Spectrometry/PTM to define the covalent structures of substances by
• 5 lectures on mass spectrometry, structural ionizing, separating and detecting molecular and
elucidation and PTM’s (post translational fragment ions according to their mass-to-charge ratios
modifications) (m/z).
• Structure elucidation practical and problems
• Practical write up assessed So, MassSpec looks at 2 characteristics of ions – mass
• Questions on exam paper + MCQ questions and charge.
Mass Spectrometry/ Structure Elucidation Lectures Advantages:
• Introduction - basic principles MassSpec is extremely sensitive and so can be carried
• How ions are made – EI, ES, MALDI out on very tiny amounts of material eg femtomoles
• How ions are separated for analysis – quadrupole, or less (10-15 mole) and can be used to study very
time of flight, ion trap, and orbitrap complex mixtures eg urine extracts, perfumes, protein
• Appearance of mass spectra – types of molecular digests etc. it is a LOT more sensitive than NMR.
ions, fragment ions,
• Collisional activation – fragment ion formation in It can also be used to study mixtures of sample. So
soft ionisation mass spectrometry (CAD-MS/MS); you don’t have to have highly purified, homogenous
the triple quadrupole and the Q-TOF samples to get good quality data, unlike Xray
• Sequencing peptides – fragmentation pathways; Crystallography. You do not need to purify proteins.
interpreting spectra
• Introduction to proteomics – MALDI mass The three basic parts of a mass spectrometer are:
fingerprinting and nanoES sequencing on the Q-
TOF and MALDI TOF-TOF
• Protein PTM’s and their analysis
Learning Objectives for all MassSpec Lectures In MassSpec, we analyze gas phase ions but the vast
• Be able to describe and compare how ions are majority of biological molecules do not exist as gas
produced, separated and detected in mass phase ions so we need to convert the biological
spectrometry material into gas phase ions and this occurs in the ion
• Be able to define peptide fragmentation pathways source. We will be looking at different methods to
and interpret simple peptide mass spectra generate gas phase ions.
• Be able to evaluate how mass spectrometry is
utilized in research and development Once we have gas phase ions, we want to separate
them based on their mass to charge ratio and this
Where to go for further information occurs in the Mass Analyzer.
• The library has a variety of books some of which
are devoted to MS, others such as “techniques” or Finally, once we have separated the ions, we need to
“methods” books often have useful chapters on detect the signals associated with them but we also
MS want to get quantitative information about which
• Search for reviews using suitable keywords – aim components of our mixture are present in a high
for mini-reviews and/or reviews in non-specialist abundance and which are present in a low
journals (Mass Spectrometry Reviews is likely to abundance.
be too advanced)
• The mass spectrometry manufacturers have some What does Mass Spectrometry data look like?
very nice videos on their websites. Most MassSpec data is expressed graphically.
• The British and American Mass Spectrometry The mass spectrum is a record of the ions that are
Society has a helpful introductory website: detected; abundance is plotted on the y-axis and the
http://www.bmss.org.uk m/z ratio on the x-axis.
http://www.asms.org
Both the intensity and mass/charge ratio do not have
specific units attached to them.
, o Also for bigger biological molecules such as
peptides, proteins, DNA, up to 500,000 Da
Electron Impact Ionization (EI) in more detail
EI ionization occurs in the gas phase so samples have
to already be in the gas phase so this is one of the
reasons why EI can only be used for smaller molecules
– larger molecules are more difficult to convert to the
gas phase.
Generally, you can create gas phase ions by heating
them up. So the sample is introduced into the source
by heating it from a probe tip until it evaporates or
The tallest peak (highest intensity) represents the from an online gas chromatograph.
most abundant component in the mixture, it has the
highest ion count. This component is then given an Because your analytical molecules are in the gas
arbitrary component of 100% relative intensity and phase, it is quite common to link the EI to a Gas
therefore all the other components in the spectrum Chromatography Separation System. So you get your
are expressed as a relative percentage of the most molecules into the gas phase and separate the
abundant peak in the spectrum. components of the mixture on an online gas
chromatography initial experiment and then do the
We can also zoom in on a specific area of the electron impact ionization and mass spectrometry
spectrum (like for example, on the high mass/charge characterization.
ratio area) if we were only interested in that area.
Once we focus on those peaks only, the peaks then Gas phase sample is bombarded with high energy
become the 100% relative intensity and all the other electrons coming from rhenium or tungsten filament
peaks would be expressed as a relative percentage of when they are heated (energy = 70 eV).
that new 100% peak :)
The name “Electron Impact” is confusing because we
Ionization (Ion) Sources do not get a direct collision between our gas phase
Different Ionization Methods in the Ion Source molecules and the electrons. Electrons are
Here are the 3 most common methods: infinitesimally smaller than the molecules so the
• Electron Impact (EI) chances of the electrons hitting our molecule is very,
o Described as a “hard” ionization technique very small. What actually happens is the high energy
which means it has high energy. Because of electrons come into close enough proximity with the
this, there can be an excess of energy given gas phase molecules so that the electron in the outer
to the biological molecules. orbital of our biological molecules is repelled by the
o Once the sample is ionized, additional energy negatively charged electron beam out of the orbital.
can then lead to fragmentation of the gas This loss of electrons from the outer orbital results in
phase ions which is useful for structural a positively-charged cation with an unpaired electron
elucidation. in its outer orbital. Hence, this cation is now a free
o Good at ionizing small molecules, 1-1000 Da radical.
but it has the lowest ionization cutoff
To summarize, ionization occurs by loss of an electron
• Electrospray Ionization (ES or ESI – Soft) to give singly-positive charged M+. (radical cation). The
o “Soft” ionization technique because enough + and dot in M+. represents a single positive charge
energy is provided to the biological molecule and the unpaired electron respectively.
in order to convert it to gas phase ions and so
there is no excess energy leading to Like we’ve said before, EI leads to fragmentation. The
fragmentation. reason for this is that the electron beam in EI is about
o For larger biological molecules such as 70eV. The average bond in a biological molecule is
peptides, oligosaccharides, proteins. For about 5eV so there is a large amount of excess energy
molecules greater than 500,000 Da associated with the electron beam. This excess energy
fragments bonds. Most of the molecular ions
• Matrix Assisted Laser Desorption Ionisation decompose into fragments (70 eV >> 5 eV bonds) via
(MALDI) uni-molecular reactions.
o Soft ionization technique
, Schematic Diagram of EI Source Ok basically, Laser energy targeted to metal target ->
First, your gas phase sample enters into the EI source. energy absorbed by matrix -> energy transfer to
There is a heated filament that produces the electron biological sample -> sample is ionized and gas phase
beam and magnets on either side to focus the beam ions are produced.
into a tight band, thus increasing the chances of
ionization of the biological sample.
Once the gas phase actually become gas phase IONS
(M+.), you can control the movements of these ions.
At the back of the EI source, you have an “Ion
repeller” with a large positive potential that will repel
the positively charged cation M+. and lead them to
move towards the MassSpec for future analysis.
The exact chemical mechanism of this ionization
process is still not well understood but it’s believed to
be similar to “flash evaporation”. We do know that we
get both positively and negatively charged ions.
However, we can only analyze one type of ion at a
time in a MassSpec.
If your sample is positively charged, you would put a
very large positive potential at the back of your target
to repel the sample into your mass spectrometer. If
your sample is negatively charged, you would put a
Note that molecular ions are radicals negatively potential to repel the sample into your
mass spectrometer.
Matrix Assisted Laser Desorption Ionization (MALDI)
in more detail Lasers for MALDI
MALDI occurs from the solid phase instead of the gas
phase. Our sample of interest is loaded on to a low
molecular weight UV absorbing “crystalline” matrix to
dry out and crystallize so you get co-crystallization of
your sample and matrix onto a metal target. This
matrix is chosen to have an absorption maximum near
the wavelength of the pulsed laser that is used to
ionise the sample.
Once you get crystals in the metal target, you Solid-state UV lasers are favored and are newer. Nd-
introduce these into the MALDI ionization source. YAG lasers generates a laser beam with a wavelength
Like most MassSpec, the MALDI Ionization Source is around 355nm whereas the nitrogen gas UV laser
under a vacuum to help the production of ions. Once generates a beam with a wavelength around 337nm.
in the ionization source, the input of energy to cause Solid-State UV Laser gives a higher energy output and
ionization comes from pulses of laser energy. Pulses faster pulsing of the lasers.
of laser energy are targeted at the crystals on the
metal target and the matrix absorbs these pulses,
resulting in enough energy being transferred to the
sample, ionizing the sample and producing gas phase
ions.
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