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제목
[MALDI-TOF] MALDI Sample Preparation Fast-Evaporation Crystallization -
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이매스 -
첨부파일
1993 년 Vorm 이란 과학자에 의해 고안된 방법으로 시료의 질량 분석기를 이용한 분석에서 보다 높은 해상도와 민감도를 얻기 위하여 보다 단순한 sample 과 matrix 를 handling 을 위한 과정을 설명하기 위한 글입니다.
Step-by-step procedure :
1. Prepare a matrix-only solution by dissolving the matrix material of choice in acetone containing 1-2% pure water or 0.1%
aqueous TFA. The concentration of matrix can range between the point of saturation or one third of that concentration.
2. Apply a 0.5 mL drop of the matrix-only solution to the sample stage. The liquid spreads quickly and the solvent evaporates almost instantaneously (1-2 sec) Note: Acetone evaporates very fast so be quick with the pipette!
3. Check the resulting matrix surface for homogeneity. Apart from a slight thickening at the edges, no inhomogeneity should be visible by light microscopy (>10X magnification). Note: If the dried spot is too big, wipe it off and reapply a smaller volume drop.
4. Apply a drop (1 mL) of sample solution (0.1-10 mM) on top of the matrix bed and allow to dry either by itself or in a flow of nitrogen. Note1: The analyte molecules can be dissolved in any solution provided that the solution does not completely redissolve the crystals (see additional comments below). Note 2: Best results are obtained when the analyte droplet is placed on the homogeneous part of the matrix and not on the rim.
5. After the drop has dried it is introduced into the mass spectrometer for analysis.
Crystal Washing: Vorm et. al. [1,2] recommend washing the crystals prior to introduction into the TOF spectrometer. A large droplet of 5-10 mL of or dilute aqueous organic acid is applied on top of the sample spot. The liquid is left on the sample for 2-10 seconds and is then shaken off or blown off with pressurized air. The procedure can be repeated once or twice. The washing liquid must be free of alkali metals and should be neutral or acidic (i.e. 0.1% TFA).
Additional comments: Analyte solutions that are basic or contain a high percentage of organic solvent can completely redissolve the matrix bed. Such damage can be easily detected with the naked eye. The problem can generally be eliminated diluting the sample with aqueous acid (0.1% or 1% aqueous TFA). Another option is to add a small droplet of 1% TFA directly on the matrix bed before adding the sample. This way the final solution is both acidic and sufficiently aqueous to dry in the normal fashion.
Pneumatic spraying: Pneumatic spraying of the matrix-only layer has been suggested as an alternative for fast evaporation [3]. The process delivers stable and long lived matrix films that can be used to precoat MALDI targets.
The fast-evaporation method provides polycrystalline surfaces with roughnesses 10-100 times smaller than equivalent dried-droplet deposits. Confocal fluorescence studies [4] demonstrated that, across an entire sample deposition area, the analyte is more uniformly distributed than with the dried-droplet method.
The improved homogeneity of the sample surface provides several advantages [5]:
(분석 시료의 표면이 보다 균질 해질수록 얻는 장점에 대하여 기술하였습니다.)
1. Faster data acquisition. All spots on the surface result in similar spectra under the same laser irradiance. No sweet-spot hunting and less averaging. The outcome of the first few laser shots is usually enough to decide the outcome of an experiment.
2. There is a better correlation between signal and analyte concentration (Still not a quantitative technique).
3. More reproducible sample-to-sample results. A good step towards better quantitation.
4. Improved sensitivity. Peptides have been detected down to the attomole level. The higher ion signals are explained as the result of the increased surface area of the smaller crystals combined with the preferential localization of the analyte molecules on the outer layers of the crystals from where the MALDI signal is believed to originate. (Note: The matrix crystals are never completely redissolved by the analyte solvent and no new matrix is added after the fast evaporation step. As a result, proteins can only be embedded on the outer layers of the pre-existing crystals.)
5. Improved washability. Salts and impurities are more easily washed off the sample deposits because the crystals are more securely bonded to the metal surface and to each other.
6. Improved resolution and mass measurement accuracy. Resolution improvements of at least a factor of two have been reported compared to dried-droplet results. The improved mass accuracy can often eliminate the need for internal standards.
7. Matrix surfaces can be prepared in advance. Precoated sample plates prepared by fast-evaporation of matrix solution on the sample spots are available from a few commercial sources.
Some of the disadvantages that have been associated with this method are:
(이 방법의 적용시에도 몇 가지 단점이 있으므로 참고를 하길 바랍니다.)
1. It does not provide reproducible sample-to-sample data for peptide and protein mixtures. If the protein or peptide sample contains more than one component, it is best to try the dried-droplet or overlayer method first. The thorough mixing of the analyte and matrix solutions prior to deposition increases the reproducibility of the spectra obtained.
2. Because the layer of protein-doped matrix on each crystal is usually very thin, it only produces ions for a few shots on a laser spot. The laser spot must constantly move to a fresh location to maintain the signal levels. This results in reduced duty cycle for the data acquisiton loop, and reduced throughput.
3. Working with very volatile solvents such as acetone makes it difficult to make reproducible sample spots. The solvent has a small surface tension and it spreads uncontrollably along the metal surface. Some varying amount of solvent is always lost to evaporation before the matrix-only droplet is delivered. The use of capillary pipette tips is recommended for handling of the liquid.
4. The method is very effective for the analysis of peptides but is not as effective for proteins [6]. The two-layer method should be tried first in the case of proteins.
References
[1] Ole Vorm, Peter Roepstorff and Matthias Mann, "Improved Resolution and very high sensitivity in MALDI TOF of matrix surfaces made by fast evaporation", Anal. Chem. 66 (1994) 3281-3287
[2] Ole Vorm and Matthias Mann, "Improved Mass Accuracy in Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of Peptides", J. Am. Soc. Mass Spectrom. 5 (1994) 955-958.
[3] H. J. Kochling and K. Biemannn, "Novel Sample Preparation for MALDI", Proceedings of the 43rd Annual ASMS Conference on Mass Spectrometry and Allied Topics; Atlanta, GA, May 21-26, 1995, p. 1225
[4] Yuqin Dai, Randy Whittal and Liang Li, "Confocal Fluorescence Microscopic Imaging for Investigating the Analyte Distribution in MALDI Matrices", Anal. Chem. 68(1996) 2494-2500.
[5] Anthony Nicola, Arkady I. Gusev, Andrew Proctor, Edwin K. Jackson and David M. Hercules, "Application of the Fast-evaporation Sample Preparation Method for Improving Quantification of Angiotensin II by MALDI", Rapid Commun. Mass Spectrom., 9 (1995) 1164-1171
[6] Yuqin Dai, Randy Whittal and Liang Li, "Two-layer Sample Preparation: A Mthod for MALDI-MS Analysis of Complex Peptide and protein Mixtures", Anal. Chem. 71 (1999) 1087-1091.