FAQs Mass Spectrometry
Q.How do I know if APAF has received my sample for Mass Spectrometry services?
A: Upon receiving samples, APAF will log
the service request and set a project code for the service requested.
APAF staff assigned to the project will send you a confirmation email,
identifying your samples, provided you have supplied a correct email
Q: When do I expect to have the result after submitting sample?
A: All samples are placed into a queue
for analysis based on sample receipt date. Please contact us prior to
sending your sample if you need you sample to be serviced urgently. For
MALDI Mass Spectrometry and 1D nanoLC ESI MS/MS analysis for protein
identification, we aim to have the report sent to you in electronic
format within 15 working days after receiving the sample. For intact
protein mass analysis, the report will be sent to you within 10 working
Non routine services generally take
longer times. Please be aware that mass spectrometers can occasionally
experience downtime that is beyond the control of APAF staff. Delays can
occur as replacement parts must be imported from overseas and fitted by
Mass Spectrometry service vendors. Depending on the type of Mass
Spectrometer service requested, some customers may experience delays in
sample processing during these instrument downtimes. In the case of any
extensive delay APAF staff will notify the customer and advise them of
the length of such delay.
Q: How do I know what service to select?
A: If you are unsure of which service to use contact Business Development on +612 98053175 begin_of_the_skype_highlighting +612 98053175 FREE end_of_the_skype_highlighting
or if you use SYYPE apafbdm. Alternatively visit the MS Services
section in this web site for detailed explanations on the mass
spectrometry services offered by APAF.
Q: How much sample is needed for the service?
A: This depends on the type of services.
|For intact protein mass analysis using ESI MS technique
1 mg of solid sample is normally sufficient. For liquid
samples the protein concentration to be no less than 5 pmol/ul and no
less than 20 ul
2D LC ESI MS/MS of complex protein samples
| 50 ug up to 200 ug of total proteins would be needed.
|For 1D LC ESI MSMS of complex protein samples
|| 2 ug up to 50 ug of total proteins would be needed.
|For protein identification from 2D gel spots
|| we recommend you cut and submit the whole spot
Provided that you can accurately detect the spot from the gel, we can
usually provide useful spectral data. Please be aware that keratin
contamination is a commonly problem seen with faint gel spots (i.e.
ratio of keratin to protein of interest is high). Take precautions when
excising gel spot. Keratin contamination can be introduced from skin
flakes, hair, dusty lab environments, contaminated excision instruments,
Q: How do I send samples to APAF?
A: You can post your sample to us in a
safe mailing envelop with the completed MS Service Request Form and
purchase order form. You may also send samples with the forms by
Dried or gel samples can be shipped at room temperature.
Liquid samples should be shipped frozen in dry ice.
If you are sending samples from overseas, it is essential that you visit
the Quarantine Guideline section on this web site and accurately follow
the quarantine instructions when sending samples.
Frequently asked questions (FAQs) related to iTRAQ®
Q. What is iTRAQ®?
A. iTRAQ® is a chemical
labelling multiplexing technique that uses mass spectrometry for
relative protein quantitation. iTRAQ is commercialised by ABSCIEX Inc.
Q. How many samples can be analysed by iTRAQ?
A. iTRAQ® is currently
available as a 8-plex kit. Because it is a relative quantitation
technique, 1 of the 8 channels is used as a reference sample to provide
relative quantitation between the other 7 channels.
Q. What should be used as the reference sample?
A. Some suggestions for the reference
sample are (i) a pooled sample made by combining an aliquot of
individual samples that will be monitored in the other channels, (ii) an
individual sample used as the reference sample. As the reference sample
is required for each iTRAQ® experiment, it is important that sufficient sample is available to complete a large iTRAQ® experiment.
Q. How much sample is required?
A. 5ug to 100ug proteins can be labelled with each iTRAQ® reagent. 50uL of plasma is required for immunodepletion and 1 iTRAQ® labelling. 1mL CSF is required for immunodepletion and 1 iTRAQ® labelling.
Q. Is immunodepletion necessary when using iTRAQ® with biofluids such as plasma?
A. Absolutely. Otherwise the number of identified proteins is small.
Q. How should the sample be prepared for iTRAQ® labelling?
A. We accept biological samples such as
cell lysate, tissues or body fluids. We also accept the extracted
proteins in solid or liquid. When sending us extracted proteins, please
make sure no primary amines are added in the samples and the salt
concentration is reduced as much as possible. Samples and the controls
need to be prepared in the same way and the total protein quantities in
the different labels need to be in the similar level. If you are to
prepare iTRAQ® labelling yourself, you should follow the instructions of the iTRAQ®
reagent kit and send us dried samples which has been labelled but not
been cleaned up using strong cation exchange chromatograph.
Q. What is APAF’s standard workflow for iTRAQ®?
A. The protein sample is solubilized,
reduced and alkylated then digested with trypsin. The tryptic peptides
are labelled with iTRAQ® reagent and mixed at a ratio so that
the protein quantities in the different labelled samples are similar.
The mixed sample is cleaned or cleaned/fractionated by SCX. Each
fraction is separated by reversed-phase gradient and injected into a
Q-Star XL mass spectrometer. The data is analysed for protein
identification and relative quantitation by ProteinPilot software (ABI)
and a report is provided to the client