Biomedical Engineering Reference
In-Depth Information
(9) The supernatant is transferred to a fresh tube and suffi-
cient 50% ACN/5% trifluoro-acetic acid to cover gel piece
is added again. This process is repeated twice.
(10) The pools of extracted peptides from each gel piece (Steps
9 and 10) are taken to dryness in a SpeedVac prior to re-
suspension in 5
l 0.1% TFA solution.
(11) Samples can be stored at
μ
20
◦
C or subject to immediate
−
mass spectrometry analysis.
3.6.Protein
Identification
byLC-MS/MS
Protein identification by mass spectrometry can be performed
using either (or a combination of) peptide mass fingerprinting
assisted laser desorbtion ionisation mass spectrometry (MALDI
MS) is fast, relatively accurate, easy to perform and is the most
commonly used technique for peptide mass fingerprinting. If
PMF fails to unambiguously identify proteins, peptide sequenc-
is generally dependant on resources and the local availability of
hardware and expertise.
(1) Prior to use the Agilent 6210 TOF mass spectrometer was
calibrated using proprietary Agilent calibration mix and
semiautomatic instrument settings in tune mode. The cali-
bration mix was directly infused into the instrument in tune
mode (via syringe pump). The updated calibration coeffi-
cients were within range and were applied.
(2) Fresh buffers for analytical nanoflow pump were prepared.
(3) An HPLC-Chip was inserted into the instrument. The
HPLC-Chip configuration included a 40 nl enrichment
column and 150 mm
×
75
μ
m analytical column packed
m materials. Prior to use the
loading and analytical pumps and lines on Agilent 1200
NanoLC system were purged for 5 min at 2.5 ml/min.
The LC stream was switched to waste and column was con-
ditioned. Base line was stable. LC stream was switched to
MS. The LC system consisted of a nanoflow pump, a cap-
illary pump for sample loading and a microwell-plate auto
sampler with cooler. Complete system control was accom-
plished using Agilent TOF LC/MS software.
(4) As samples from 2D gels are not complex a relatively short
LC gradient method can be used to introduce peptides into
(5) 0.5
with Zorbax 300SB-C18, 5
μ
l of sample was analyzed using Agilent's microfluidics-
based HPLC-Chip for nanoelectrospray LC-MS connected
to Agilent's Q-TOF MS.
(6) Instrument settings, Ion Source HPLC-chip, positive
mode with centroid data storage selected. Reference mass
μ
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