Chip Reading and Data Acquisition
The PBS IIc SELDI-TOF mass spectrometer reader is a laser desorption ionization time of flight mass spectrometer equipped with a pulsed ultraviolet nitrogen laser source. When the laser activates the bound serum biomarkers on the chip surface, the biomarkers become desorbed and ionized. Ionized molecules fly along the mass spectrometer to the ion detector in a time of flight manner according to their mass over charge ratio (m/z). When the ion signal is detected in the ion detector, signal processing is accomplished by high-speed analog-to-digital converter linking to a computer. Detected protein biomarkers are displayed in spectral, map or gel view formats by the Ciphergen ProteinChip software 3.0.2. The following steps show how the chips are read in the mass spectrometer.
Click the “Sample Exchange Dialog” button from the manu bar of the Ciphergen ProteinChip software 3.0.2 in the PBS IIc SELDI-TOF mass spectrometer reader.
Click “Open Lid” button in the “Sample Exchange Dialog” box to open the lid of the chip chamber.
Place the sample treated chips into the slot of the chip chamber.
Click “Close Lid” button.
The chip is automatically inserted into the chip chamber.
In the “Sample Exchange Dialog” box, input the serial number, chip type, and chip format.
Click “Chip” button from the “Sample Exchange Dialog” box.
Input sample name and sample group.
Click “OK” button.
Click “Chip Protocol” button from the menu bar.
Input spectrum tag (which is the sample name) and the following spot protocols: Starting laser intensity to 165.
Starting detector sensitivity to 9.
Highest mass of detection to 200,000 Da.
Optimal mass range of detection from m/z of 2000 to 20,000 (signals from m/z of 0–2000 are not analyzed as artifacts can be produced by energy-absorbing molecules or other chemical contaminants at this mass range).
Focus lag time at 800 ns.
Mass deflector to Auto.
Data acquisition method to SELDI quantitation.
338 laser shots per sample.
Click “Start Running” button from the menu bar.
By the time 338 laser shots are fired, the collected data is automatically saved.
It is important to first carry out external calibration of the equipment using the All-In-1 Peptide molecular mass standard (according to the instruction sheet from the manufacturer) to ensure mass accuracy of the spectra before running the samples.
Carry out baseline subtraction and calibration for the spectra.
Generate labeled peak groups (clusters) across multiple spectra by the Biomarker Wizard mode.
Compare peak groups detected in the SARS patients with those of the controls by nonparametric two sample Mann-Whitney U-test in the Biomarker Wizard mode.
Biomarker Wizard operates in two passes, with the first pass uses low sensitivity settings to detect obvious and well-defined peaks and the second pass uses higher sensitivity settings to search for smaller peaks.
This will identify both strong and weak peaks that are significantly increased or decreased in the patients vs the controls.