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ABSTRACT |
For MALDI-TOF-MS analysis of proteins and peptides, samples are cocrystallized with an excess of organic matrix that absorbs at a specific wavelength (usually, UV337nm). Typically, sinapinic acid (SA) is the matrix of choice for large proteins, whereas {alpha}-cyano-4-hydroxy-cinnamic acid (HCCA) is the preferred matrix for peptide mapping. Following a short laser pulse, analytes are protonated and desorbed into the gas phase, and their m/z values are determined in a TOF mass analyzer. Mass accuracy determinations vary from ±0.01% to 0.1% depending on the sample preparation technique and the method used for mass calibration.
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MATERIALS
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Reagents
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Calibration standards (1-10 pmol/µl in 60% acetonitrile containing 0.1% [v/v] TFA)
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To obtain the best interpretation of MALDI-TOF-MS data, standards of known molecular mass close to the molecular mass of the unknown sample are required to ensure a linear calibration curve. These must be prepared and chosen carefully. Typically, angiotensin I or angiotensin II, whose accurate molecular masses are known, can be used for peptide mapping studies. Table 2 and Table 3 contain a range of peptides and proteins that can be used routinely as standards in MALDI-MS analysis. All of these standards are commercially available, and therefore, stock solutions can be accurately prepared. Stock solutions of peptide calibrants are prepared at a concentration of 100 pmol/µl aliquots (frozen stocks are made up in 60% acetonitrile containing 0.1% TFA, and can be kept for several years). For working solutions, these aliquots are diluted to 1-10 pmol/µl with aqueous 0.1% (v/v) TFA. Working solutions of calibrant can be used for 1 week when kept at 4°C. The concentration of the calibration standards must be similar to that of the unknown sample (range 1-10 pmol/µl) to ensure accurate results.
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60% acetonitrile containing 0.1% [v/v] TFA
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Lyophilized samples (dissolved in 60% acetonitrile containing 0.1% [v/v] TFA at a concentration of 1-10 pmol/µl) or RP-HPLC-purified fractions, which can be used directly
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Samples containing excipients such as buffers, salts, detergents, and denaturants must be desalted prior to analysis. Even minor quantities of sodium (m/z 23) and potassium (m/z 39) ions, readily generated via laser ionization, can cause significant ion suppression.
The detergent n-octyl glucoside (0.1%) can be added at different stages of sample preparation (e.g., digestion or solubilization of digested peptides) to prevent adsorption of peptides on the sample tube wall and/or pipette tip, thereby yielding increased peptide peak number and improved sequence coverage.
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MALDI-MS matrix solution
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20 mg/ml matrix (e.g., HCCA, DHB, or SA)
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60% acetonitrile
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0.1% (v/v) TFA
Under these conditions, the matrices are saturated solutions and must be centrifuged prior to use.
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Methanol
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Equipment
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Lint-free tissues (e.g., Kimwipes)
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MALDI mass spectrometer
See the note to Step 6 for more information.
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MALDI plate
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Pipette tips, 2-µl
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Oven, 40°C (optional; see Step 3)
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METHOD
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Pipette 0.5 µl of the MALDI-MS matrix solution (using 2-µl pipette tips for accuracy) onto the sample wells of the metal sample plates used for MALDI-MS analysis.
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Immediately add 0.5 µl of the standard or sample to the matrix before it dries.
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Allow the solvent to evaporate and the samples to dry for ~5 minutes either at room temperature or in a 40°C oven.
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Transfer the metal sample plate to the vacuum chamber of the mass spectrometer.
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Acquire an initial mass spectrum at a laser power well above the ionization threshold to "warm up" the calibration standard.
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Decrease the laser power until a good spectrum is obtained.
Steps 5 and 6 apply to MALDI-TOF instruments that are equipped with a high-vacuum ionization source (e.g., Kompact MALDI IVTM; Kratos Analytical/Shimadzu, or equivalent). For instruments equipped with a CCD camera for viewing the cocrystallized sample (e.g., o-MALDI QSTAR Pulsar I; Applied Biosystems, or equivalent), select a large crystal for analysis (e.g., large crystals form when using the matrix DHB; this is not so important when using HCCA or SA, which usually yield uniformly small crystals). HCCA is preferred as a MALDI matrix for peptide mapping because it yields the highest sensitivity and forms a uniform matrix layer on a MALDI sample plate, which makes it amenable for automated analysis. For optimal results, run the unknown samples at approximately the same laser power as the calibration standard. Although a high laser power may give a good signal, resolution may be compromised due to peak broadening. The use of a low laser power at the ionization threshold may give high resolution, but result in poor signal-to-noise ratios. Typically, the thresholds of ionization for HCCA and DHB are 20 and 30 µJ, respectively. Thus, HCCA is considered to be a "hotter" matrix than DHB, which gives rise to increased metastable ion formation and concomitant PSD. A consequence of the latter is broader peak formation and reduced resolution. Although a sample may appear to be uniform, it is recommended that different regions of the spot be examined to find "sweet spots," that is, regions of the sample spot that give superior signal-to-noise ratios.
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Obtain a linear external two-point calibration using an appropriate calibrant.
When using HCCA, the matrix-derived ion (e.g., [M+H]+-OH, m/z 173.2) and the singly charged ion of the appropriate calibration standard can also be used as calibrants
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Repeat Steps 5 and 6 for unknown samples and compare with the calibration values to obtain accurate masses.
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After use, clean the MALDI plate by rinsing thoroughly with H2 O to remove any visible crystals and then with methanol; wipe with lint-free tissues (e.g., Kimwipes).
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REFERENCES
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Kussmann, M. and Roepstorff, P. 2000. Sample preparation techniques for peptides and proteins analyzed by MALDI-MS. Methods Mol Biol 146: 405-424.[Medline]
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Anyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in this protocol and has no liability in connection with the use of these materials. Materials used in this protocol may be considered hazardous and should be used with caution. For a full listing of cautions regarding these material, please consult:
Proteins and Proteomics, A Laboratory Manual, by Richard J. Simpson, © 2003 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 478-481. |
