Dit document omvat de info van de slides plus mijn lesnotities van les 6
van concepts, gegeven door Xaveer Van Ostade. De lessen van Kurt Boonen (Les 2,3,4 en 8) heb ik ook, maar de notities staan op de slides. Moest je hierin ook geïnteresseerd zijn, mag je me altijd contacteren via messenger :...
Les 6: 8/11
Mass spectrometry in proteomics – instruments
Introduc)on
- Molecules from a sample are ionised and converted to the gas phase, followed by
separa:on according to their mass/charge (m/z) ra:o (not only mass!)
o Then you can play around it, and you can easily direct it in vacuum
- Hence, aBer ionisa:on of a molecule by the addi:on of H+ ions, the m/z ra:o of the
molecule is measured. The same holds for the opposite, when H+ is removed.
o It measures the mass and the charge and the ra:o of these two is given in the
spectra
- Essen:al steps are:
o Produc:on of ions in the gas phase
o Accelera:on of ions in an electric field
o Separa:on of ions by a mass analyser
o Detec:on of each ion with a specific m/z ra:o
- The instrument has a high vacuum: 10-6 Torr
o To perform correct measurements
- Finally, spectra are being processed by a data system
Some essen)al terms
Mass-charge ra+o
- Ions are being produced by the addi:on of one or more protons.
- This means that a pep:de of MW 2000 Da has a m/z value of:
o 2001/1 = 2001 aBer addi:on of 1 proton ([M+H]+)
o 2002/2 = 1001 aBer addi:on of 2 protons ([M+H]2+)
o 2003/3 = 667,67 aBer addi:on of 3 protons ([M+H]3+)
o etc…
- The same pep:de can have different numbers of charges.
Resolu+on
- The smaller the peaks to more informa:on you have -> more sensi:ve
- The ability to separate and measure masses of ions with similar (but not iden:cal) MW.
The resolu:on is dependent on the shape of the peak.
- Defined as m/Δm in two ways:
o 10% valley defini:on: two successive ions with mass m1 and m2 are posi:oned
such that between them is a ‘valley’ of 10%, rela:ve to the height of the least
abundant ion. The difference between m1 and m2 is Δm and the resolu:on is
m2/Δm (m2/Δm2 in figure).
§ You searching for pep:des that overlap each other of 10% of the height of the
smallest peak
§ Different between m2 and m1 is the delta m and the resolu:on is m2/delta m
-> the higher delta m is the beaer the resolu:on
o ‘Full-width, half maximum’ (FWHM): the width of one ion peak with mass m at 50%
height is Δm. Then FWHM = m/Δm (m1/Δm1 in the figure).
§ Looks at one peak
§ Most manufactures use the full width maximum
, - Resolu:on is constant over the m/z range -> peak widths increase geometrically with
increasing mass!
o If the peaks are going to the right, m is increasing -> delta m must also inccrease in
order to have a constant resolu:on -> peakes become more wider
- A high resolu:on (>10.000) is necessary for MW determina:on of pep:des with MW
of 1000-5000 Da.
- The higher the resolu:on the much beaer the measurement will be
- Peak separa:on is strongly dependent on the instrument resolu:on:
Monoisotopic mass vs. average mass
- Two important carbon isotopes: 12C and 13C with resp. masses of 12,000000 and
- 13,003355 Da and with abundancy of 98.92 and 1.08%, resp..
- Two important nitrogen isotopes: 14N and 15N with resp. masses of 14,003074 and
15,000108 Da and with abundancy of 99.63 and 0.37%, resp..
- Lot of nitrogens and carbons integrated in the pep:des/proteins -> certain frac:on of
this higher weighted isotopes inserted in the pep:des/proteins -> we need to be aware
of this
- Monoisotopic mass of an analyte is the sum of all monoisotopic masses of all elements
in that analyte.
o Mass of the pep:de that only contains the light isotopes
- In prac:ce, a certain percentage of heavier isotopes is present in the analyte, especially
when the analyte is of high MW. All these isotopic analyte variants together are called
an ‘isotope cluster’ which can be seen in mass spectrometers with a high resolu:on.
o One have one heavy isotope, other maybe 5
- The chemical average mass of an element is the average mass of all isotopes with due
of their abundancy. For instance, 98.9% 12C and 1.1% 13C give a chemical average mass
of 12.011 Da.
- Hence, the (chemical) average mass of an analyte is the sum of the chemical average
masses of all the elements within the analyte. This average is theore:cal and does not
necessarily correspond with a peak.
- The peak top mass of an analyte is the mass that corresponds with the highest peak of
an ioncluster.
- Hence:
o Small pep:des: monoisotopic mass corresponds more or less with the peak top
mass, however this correspondance disappears when pep:des become larger
§ Angiotensine
• Higher resolu:on of 10 000 -> 4-5 peaks
, • The peak on the leB is the monoisotopic peak -> no heavy elements are
incorporated.
• But some pieces have 1 of more heavy isotopes incorporated -> isotope
cluster
• Average chemical mass is slightly more to the right -> takes the heavy
isotopes in considera:on
o For larger proteins the peak top mass corresponds with the average mass.
Distribu:on of the different isotopic peaks becomes more Gaussian as the MW
increases.
§ Insuline
• Slightly larger MW
• The peak top shiBs a liale bit to the right -> we have more molecules that
have incorporated one heavy isotope.
• The more the pep:des grows the peak top mass will shiB more to the right
• Average mass falls together with the peak top mass
Accurarcy
- Measure for the difference, seen between theore:cal m/z and measured m/z.
Dependent on stability and resolu:on of the instrument.
- Wriaen as percentage (e.g. mass= 1000±0.01%), or as parts per million
(1000±100ppm).
- Increasing MW-> fault on accuracy will increase correspondingly.
- Example: accuracy of 0.01% (or 100ppm) corresponds to a variability of:
o 0.1 Da with an m/z value of 1000
o 0.5 Da with an m/z value of 5000
o 5 Da with an m/z value of 50.000
o Etc…
Ionisa)on
Introduc+on
- Posi:ve as well as nega:ve charges can be produced. Principles of separa:on and
detec:on are the same, but posi:ve charges are mostly used.
o Posi:vely charges are the most used in proteomics
- Different methods were applied on proteins in the past, with limited succes rates.
o Electron impact (EI):
§ Electron current (produced by heated metal) collides with high speed on the
analyte in gas phase -> analyte takes up or loses an electron (becomes anion
or ca:on, resp.)
§ Mostly applied on organic compounds since proteins/pep:des cannot be
converted to the gas phase.
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