LC-MALDI and MALDI-MS/MS for the Analysis of Complex Peptide Mixtures
Joseph Fox1, Joanne B. Connolly2, Matthew E. Openshaw2
1Shimadzu Scientifi c Inc., Columbia, US; 2Shimadzu Biotech/Kratos, UK
LC-MALDI provides several advantages over conventional single spot (i.e. no separation) acquisitions for the analysis of complex peptide mixtures. These include (i) concentration and separation of the peptides prior to analysis, (ii) extended time available for MS/MS interrogation, (iii) separation of isobaric peptides resulting in increased sequence coverage. The ability to separate and concentrate the peptides prior to analysis results in higher sensitivity and means that more MS/MS spectra can be generated from the sample. Rather than acquiring all the MS/MS spectra from the same spot, with the sample being depleted with each MS/MS spectrum acquired, MS/MS spectra are generated from different positions on the MALDI target and sample depletion is less of an issue.
In the LC-MALDI technique, the protein mixture is enzymatically digested and the resulting peptides are separated
prior to MALDI analysis (see Figure 1). Automated MALDI time-of-fl ight (TOF) mass spectrometric (MS) analysis is
used to select suitable candidates for subsequent MS/MS fragmentation and proteins are identifi ed using the MS/MS
data obtained for the individual peptides. We present results obtained using a new MALDI-TOF mass spectrometer,
capable of true high-energy MS/MS (AXIMA-TOF2), demonstrating the utility of this technique for the analysis of
complex protein mixtures. Also presented is data obtained for an iTRAQ™ labelled protein mixture, including a novel
MALDI-MS/MS analysis mode (Low Mass Zoom (LMZ)) which can be used to enhance fragment ion resolution for low
mass fragment ions, e.g. diagnostic ions used for relative quantifi cation (iTRAQ™).
Protein Mixture: A 50 protein mixture (5 pmol of
each protein) was digested using trypsin. 5 uL of
the digest supernatent (500 fmol per protein) was
loaded onto a 300 um i.d. x 15 cm C18 column (LC
Packings) and the peptides were separated using a
linear acetonitrile/water/TFA gradient (~5 µL/min).
The LC eluent was automatically deposited onto an
unmodifi ed stainless steel MALDI target using the
AccuSpot MALDI spotting robot (Shimadzu, Japan).
Fractions were collected every 6 sec from T = 3
- 41 min. MALDI matrix solution (CHCA; 5 mg/mL in
50/50 acetonitrile/0.1% TFA) was added and mixed
with the LC eluent using the AccuSpot.
iTRAQ sample: 2 samples of an equimolar 5-protein
mixture were labelled using the 114 and 117
reagents using the manufacturers recommended procedure. The two samples were combined and prepared for LCMALDI
analysis using an SCX trap. 5 µL of the digest mixture (500 fmol per protein) was loaded onto a C18 column
and prepared as described above.
MALDI-MS and MALDI-MS/MS acquisitions were performed on an AXIMA-TOF2™ mass spectrometer (Shimadzu
Biotech/Kratos, UK). All acquisitions were performed in fully automated mode using the LC-MALDI software
functionality of the MALDI-MS software. The minimum precursor intensity threshold was set to 30 mV and there was
no limit on the number of candidates selected for MS/MS (i.e. all precursors meeting the acceptance criteria were
selected). For MS/MS acquisitions, helium was used as the collision gas.
Protein Mixture Sample
Using the LC-MALDI technique described, we successfully identifi ed 33 proteins from the 50 protein mixture.
Additionally, 4 other proteins were also identifi ed which were not intentionally supposed to be present in the sample.
It is believed that the use of multiple (complementary) mass spectrometric techniques, e.g. electrospray LC-MS/MS,
would result in the identifi cation of even more proteins. These results are very encouraging and demonstrate the
potential of the LC-MALDI technique combined with MALDI-MS/MS for the analysis of protein mixtures.
iTRAQ Labelled Sample
Results obtained using a 5-protein test mix demonstrated that the AXIMA-TOF2 can be used for the relative
quantifi cation of proteins using the iTRAQ labelling technology. Using the 114 and 117 reagents, the fragment ion
resolution obtained in MS/MS mode is suffi cient to allow the determination of relative expression levels between
samples with high precision (±15%). For higher mass precursor ions, a novel MALDI-MS/MS analysis mode (LMZ) can
be used to enhance the fragment ion resolution and sensitivity of the low mass fragment ion region containing the
iTRAQ reporter ions. It should be stated that the LMZ mode is often not necessary for lower mass precursors as the
fragment ion resolution obtained at lower mass in full MS/MS mode is suffi cient for the quantifi cation calculations.
• LC-MALDI is a suitable technique for the automated analysis of complex protein mixtures.
• When coupled with the AXIMA-TOF2 MALDI-TOF mass spectrometer, the high-energy MS/MS provides high
confi dence identifi cations.
• Relative quantifi cation can be achieved using the iTRAQ™ technology. A novel MS/MS mode (Low Mass Zoom) can
be used, if required, to enhance the resolution and signal intensity of low mass fragment ions.
iTRAQ is a registered trademark of Applied Biosystems
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