Lecture 9: 12/12
LC-MS
- HPLC: beat smaller diameter -> pu5 larger pressure on the column so that the solvent
goes through the column
- Separa=on of complex mixtures of proteins and pep=des, immediately followed by
mass spectrometric analysis of the separated components.
- Online connec=on of chromatography to the ESI source: eluted and separated pep=des
are directly sent into the mass spectrometer where the MW (MS scan) and the AA
sequence (MS/MS scan) are determined.
- 1 duty cycle is the =me the MS needs to perform 1 MS scan and several MS/MS scans
of the highest peaks (see below)
- You can direct elute the elusion in the ESI source
o You don’t lose sample
- If we have complex mixtures of pep=des and proteins -> you elute several pep=des
simultaneously from the column -> MS cannot follow
- Eluted pep=des are directly introduced in the MS -> MS scan and MS-MS scan
- Since an MS/MS scan demands some =me and chromatography runs con=nuously, a
selec=on must be made of the ions (precursors) that have to undergo an MS/MS scan:
o In prac=ce the mass spec runs 1 MS scan (± 1 sec.) allowing to determine the
pep=de masses within a certain =me slot, followed by an MS/MS scan on three
to eight selected (usually most intense) pep=de (total ± 5 sec.). Total = 1 duty
cycle.
§ Now: ms
§ More sensi=ve
§ You will pick up more pep=des for one protein-> reliability of the
iden=fica=on increases dras=cally
o If sample is very complex: several pep=des reside within one chromatographic
peak and cannot always be detected and sequenced by the mass spectrometer
(peak satura=on).
§ To reduce the peak satura=on, we can tell the MS that if it sees a pep=de
with a m/z value of 837,5 don’t select it because it comes for example
of kera=n
-> Therefore, exclusion lists, containing pep=des that can be neglected (no
MS/MS scan), can be entered into the mass spectrometer.
- Usually: reverse phase chromatography at micro or capillary scale. If chromatographic
flow (5-1000 µl/min) is too high for the mass spec (=ll 500 nl/min.) a ‘spli5er’ (part to
waster and part to ESI source) is used
o Nanoscale is for the analy=cal scale
- Nanopumps with a reproducible linear speed =ll ± 50 nl/min. are also available.
o Nano scale has several advantages:
§ Lower flow rates (see above) are similar to these from ESI source, hence no
spli5er needed and less sample and solvent go to the waste.
§ Nano column can be used as ionisa=on source
§ Increased sensi=vity since the volume solvent per =me unit is reduced,
allowing the sample to concentrate.
LC-MS
- HPLC: beat smaller diameter -> pu5 larger pressure on the column so that the solvent
goes through the column
- Separa=on of complex mixtures of proteins and pep=des, immediately followed by
mass spectrometric analysis of the separated components.
- Online connec=on of chromatography to the ESI source: eluted and separated pep=des
are directly sent into the mass spectrometer where the MW (MS scan) and the AA
sequence (MS/MS scan) are determined.
- 1 duty cycle is the =me the MS needs to perform 1 MS scan and several MS/MS scans
of the highest peaks (see below)
- You can direct elute the elusion in the ESI source
o You don’t lose sample
- If we have complex mixtures of pep=des and proteins -> you elute several pep=des
simultaneously from the column -> MS cannot follow
- Eluted pep=des are directly introduced in the MS -> MS scan and MS-MS scan
- Since an MS/MS scan demands some =me and chromatography runs con=nuously, a
selec=on must be made of the ions (precursors) that have to undergo an MS/MS scan:
o In prac=ce the mass spec runs 1 MS scan (± 1 sec.) allowing to determine the
pep=de masses within a certain =me slot, followed by an MS/MS scan on three
to eight selected (usually most intense) pep=de (total ± 5 sec.). Total = 1 duty
cycle.
§ Now: ms
§ More sensi=ve
§ You will pick up more pep=des for one protein-> reliability of the
iden=fica=on increases dras=cally
o If sample is very complex: several pep=des reside within one chromatographic
peak and cannot always be detected and sequenced by the mass spectrometer
(peak satura=on).
§ To reduce the peak satura=on, we can tell the MS that if it sees a pep=de
with a m/z value of 837,5 don’t select it because it comes for example
of kera=n
-> Therefore, exclusion lists, containing pep=des that can be neglected (no
MS/MS scan), can be entered into the mass spectrometer.
- Usually: reverse phase chromatography at micro or capillary scale. If chromatographic
flow (5-1000 µl/min) is too high for the mass spec (=ll 500 nl/min.) a ‘spli5er’ (part to
waster and part to ESI source) is used
o Nanoscale is for the analy=cal scale
- Nanopumps with a reproducible linear speed =ll ± 50 nl/min. are also available.
o Nano scale has several advantages:
§ Lower flow rates (see above) are similar to these from ESI source, hence no
spli5er needed and less sample and solvent go to the waste.
§ Nano column can be used as ionisa=on source
§ Increased sensi=vity since the volume solvent per =me unit is reduced,
allowing the sample to concentrate.
