|Home | About | Journals | Submit | Contact Us | Français|
A new method for the mass calibration of the matrix-assisted laser desorption/ionization-mass spectrometry spectrum is introduced. This method achieves the same accuracy as that of internal calibration but without its drawbacks. The interference and signal suppression by calibration standard are avoided, and a pure/clean sample spectrum is obtained. No prior knowledge about the sample quantity is required for the calibration. The effectiveness of the method is demonstrated with protein identification data.
Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) is an important technique in analyzing proteins and other molecules.1–7 It usually contains a time-of-flight mass analyzer for the measurement of molecular mass. In the mass spectrometer, the molecule, mixed with the matrix, is ionized by the laser and flies to the detector. The time of the flight is converted into the molecular mass for the measurement, which is usually calibrated with standard molecules of known masses.8–14
There are two basic types of calibration methods: external and internal.8–14 For the external calibration, a standard sample is put in one or more spots, and the calibration parameters are obtained from this standard sample.8–11 These calibration parameters are used to calibrate samples in other spots. For the internal calibration, the standard molecules are mixed with the sample, and a MALDI-MS spectrum of the mixture is acquired.8,12–14 The peaks of the standard molecules are identified, and masses of the standard molecules are used to calibrate the entire spectrum. The routine external calibration is convenient but often not accurate, especially for MALDI mass spectrometers of early models.8 The internal calibration could be accurate but has the following drawbacks.8 A prior knowledge of the sample quantity is needed to determine how much standard molecule should be added to the sample for the internal calibration. If the standard molecule quantities are insufficient, the standard MS peaks may not be identified for the calibration. If too many standard molecules are added, the sample MS peaks will be suppressed. The standard molecule MS peaks may overlap with the sample MS peaks. Unexpected or unknown molecules in the standard sample also add interfering MS peaks to the spectrum. The internal calibration can also be performed using matrix or enzyme autolysis MS peaks. However, these peaks are suppressed when the sample signal is strong.
Here, a new calibration method is introduced, which is accurate like the internal calibration but avoids its drawbacks. An accurate MALDI-MS spectrum can be obtained without additional MS peaks from the calibration standard, and other interfering molecules and no prior knowledge of the sample quantity are needed. The method is demonstrated with protein identification data.
The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA): alcohol dehydrogenase (ADH; from horse liver, Cat. #A9589), O-methylisourea hemisulfate salt (Cat. #455598), DTT (Cat. #43815), iodoacetamide (Cat. #I1149), ammonium bicarbonate (ABC; Cat. #A6141), and trifluoroacetic acid (TFA; Cat. #T62200). Trypsin (Trypsin Gold; Cat. #V5280) was from Promega (Madison, WI, USA). α-Cyano-4-hydroxycinnamic acid (CHCA; Cat. #186002331) was from Waters (Milford, MA, USA). Hydrochloric acid (Cat. #9535) was from J.T. Baker (Phillipsburg, NJ, USA). Calmix (4700 Proteomics analyzer calibration mixture) was from Applied Biosystems (AB; Foster City, CA, USA).
ADH (30 pmol) was isolated in a SDS-PAGE gel band at 40 kD. The gel band was excised into small pieces (~1.5 mm diameter) and destained completely with the destain solution containing 0.1 M ABC and 50% acetonitrile (ACN). The wet gel pieces were dehydrated with ACN and dried under vacuum in a SpeedVac. The dry gel pieces were impregnated and covered with the digestion solution containing 10 ng/μl trypsin, 10% ACN, and 40 mM ABC. The sample was incubated at 37°C overnight. The peptides were extracted with the extraction solution containing 50% ACN and 0.1% TFA. The solution was dried under vacuum in a SpeedVac. To guanidinate the peptides, 30 μl guanidination solution (containing 1 M O-methylisourea hemisulfate adjusted to pH 11.0 with NaOH) was added to the sample, and the sample was Votexed to dissolve the peptides. The sample was incubated at 37°C overnight. Then, 1.5 μl TFA was added to the sample to make the sample pH <3. ZipTip (with 0.2 μL C18 resin) and the matrix solution containing 2.5 mg/ml CHCA, 50% ACN, and 0.1% TFA were used for eluting and spotting the samples onto the MALDI sample plate.
A VOYAGER DE-PRO MALDI mass spectrometer from AB was used for the study. The MS function in reflector modes for positive ions were applied. The MALDI-MS spectral data were processed using the Data Explorer software from AB. The peptide monoisotopic peak list was verified manually. The Mascot software from Matrix Science (Boston, MA, USA) was used for the protein identification in peptide mass fingerprint mode using the peptide monoisotopic peak list.
The new MALDI-MS data acquisition and calibration method include the following five steps.
Figure 1 shows the spectra of the ADH sample. After the acquisition of the spectrum S1, approximately 100 fmol Calmix was added to the sample spot for the acquisition of the spectrum S2. The calculated Calmix standard peptide ion monoisotopic masses are 904.4681, 1296.6853, 1570.6774, 2093.0867, 2465.1989, and 3657.9294. These standard peptide MS peaks appear in S2 and are absent in S1. They can be identified in S2 at observed masses 904.90, 1297.14, 1571.13, 2093.51, 2465.53, and 3658.08. S2 was then calibrated to obtain S2_cal using the internal calibration feature of Data Explorer and these standard Calmix peptide peaks.
The sample peaks at 880.4624, 1210.6912, 1653.9025, 1952.1184, and 2987.5129 (monoisotopic masses) in S2_cal were selected as standard sample peptide MS peaks. These peaks and masses were used to calibrate S1. The corresponding standard sample MS peaks in S1 can be identified at 880.90, 1211.15, 1654.35, 1952.56, and 2988.87, respectively. These peaks and their calibrated masses from S2_cal were used to calibrate S1 using the internal calibration function of Data Explorer, and the calibrated sample spectrum is S1_cal.
S1_cal is internally calibrated but does not contain additional MS peaks from the calibration standard Calmix. The monoisotopic peak list was generated from S1_cal and used for the identification of the protein using the peptide mass fingerprint method. The ADH protein (gi126723445) was identified in the NCBInr database with a high Mascot score of 152. The identified peptides and their mass measurement deviation (S1_cal Mo error) in ppm are listed in Table 1. The general mass deviation is within 20 ppm, which is very good for protein identification.
The protein identification was also performed using the MS peak list generated from S1, but the ADH protein could not be identified from the NCBInr database with a sufficient Mascot score. The observed masses of S1 spectral peaks corresponding to identified peptides in Table 1 were found to be 101–505 ppm (S1 Mo error in Table 1), deviated from the actual/calculated peptide masses. This shows that a dramatic improvement in mass accuracy was achieved by the calibration in S1_cal.
The spectral and protein identification data shown above demonstrated the effectiveness and application of the new MALDI-MS calibration method. This method does not require prior knowledge about the sample quantity. The calibration accuracy of this method is as good as that from standard internal calibration. This method is clean, as a pure or clean sample spectrum S1_cal is obtained without additional MS peaks from calibration standard peptides. It does not have the problem of sample MS peak suppression by the calibration standard.
We thank Susan Macisaac and Keith Cromack at Monsanto for their support.