Protein ID Solutions Sample Prep Guide
109
Familiarization, Checkout, and Troubleshooting for the Overall Solution
10
Problem with digestion
or recovery from gel
bands
See the troubleshooting section in the appropriate chapter on digestion.
·
If you did in-gel digestion, see
"Troubleshooting"
on page 63.
·
If you did in-solution digestion, see
"Troubleshooting"
on page 77.
Desalting problem
Make sure you followed the protocol exactly.
·
If you used Cleanup C18 Pipette Tips, see
"Protocol for sample
cleanup"
on page 83.
·
If you used Cleanup C18 Spin Tubes, see
"Protocol for sample
cleanup"
on page 93.
Injection problem
· Check for an air bubble in bottom of vial and tap vial gently to
dislodge.
· Check that you used the correct vial type. The injection offsets listed
in the protocols apply only to the vial types specified there.
· To check that the correct volume is being injected, place a known
volume of water into a vial and measure the volume after an injection.
Problem with
conventional nanoflow
LC/MS/MS or
HPLC-Chip/MS system
To check the system, do one of the following:
·
Dilute HSA sample 1:20 in water, perform in-solution digestion, and
analyze the digest. See instructions in this chapter.
·
Analyze a standard digest such as BSA from a reputable
manufacturer.
Bad chromatography
Leaks
Find and correct leaks.
Wrong mobile phases
Prepare the mobile phases specified in Chapter 2 or 4 of the LC/MS
Application Guide.
Failed digestion
See the troubleshooting section in the appropriate chapter on digestion.
·
If you did in-gel digestion, see
"Troubleshooting"
on page 63.
·
If you did in-solution digestion, see
"Troubleshooting"
on page 77.
Some other LC problem
See troubleshooting information in the manuals you received with the
LC modules.
Peaks present in
chromatogram, but
protein not identified
as HSA.
Ion trap mass axis not
properly calibrated
Check mass axis calibration (Calibration tab in MSD Trap Control).
No MS/MS done.
Check MS/MS threshold (MS(n) tab in MSD Trap Control) and adjust
for your system.
Peaks present in
chromatogram, but
signal is low.
New column or
HPLC-Chip installed, but
not pre-conditioned
After you install a new column or HPLC-Chip, make two injections of a
high-level sample (e.g., 500 fmol BSA digest), followed by three blank
injections.
Problem
Cause
Solution/how to check