Non-porous membrane for MALDI-TOFMS

Details Non-porous membrane for MALDI-TOFMS Non-porous membrane for MALDI-TOFMS

1999-01-29

2001-07-24

Berman, Jack (Department: 2881)

Radiant energy

Ionic separation or analysis

With sample supply means

C250S287000

Reexamination Certificate

active

06265715

ABSTRACT:

The present invention relates to a non-porous membrane for use as a sample support in matrix-assisted laser desorption time-of-flight mass spectrometry.
BACKGROUND OF THE INVENTION
Within the last decade, with the advent of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), mass spectrometry has been shown to be able to contribute to the rapid, sensitive and accurate characterization of biomolecules. These techniques have allowed for the development of mass spectrometry-based methods for investigation of biomolecular structure and function. However, in order to achieve the best analysis, these techniques must be performed quickly, accurately and with a minimum of sample loss.
For example, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) (Karas et al., 1991,
Mass Spectrom. Rev.
10:335, Hillenkamp and Karas, 1990,
Meth. Enzym.
193:280, Zaluzec et al., 1995,
Prot. Exp. Purifification
6:109) provides a rapid and convenient means for the characterization of proteins and peptides derived from biological samples. This method has the advantage of being relatively tolerant of impurities, such as salts and buffers. As a result, molecular ions of peptides and proteins can still be produced by MALDI, even with salts or buffers at concentrations which would hamper other ionization processes such as electrospray ionization (ESI). Delayed extraction, combined with reflecting time-of-flight mass analysis, provides high resolution and allows accurate mass measurements for sample components in the parts-per-million range, even for fairly complex mixtures (Vestal et al., 1995,
Rapid Commun. Mass Spectrom.
9:1044). In spite of the relative tolerance to impurities mentioned above, biologically derived samples must still be isolated and purified prior to analysis to obtain the best results. Specifically, MALDI analysis of complex mixtures is often hindered by the suppression or quenching of the signals from some analytes. In addition, the presence of impurities may inhibit the formation of matrix-analyte crystals suitable for the MALDI ionization process. The impurities may also lead to the formation of adducts which will degrade the resolution and mass accuracy of the results. Typically, this is overcome by purification of the sample prior to analysis.
Several methods of sample purification prior to MALDI-TOFMS analysis have been developed including dialysis and chromatography. Both methods have limitations, such as sample loss and time-consuming sample preparation. Alternatively, the desired analytes may be removed from the sample using affinity binding-based purification methods. Specifically, these methods selectively retain and concentrate the analytes of interest; however, these methods are time-consuming which may in turn result in degradation of the target analytes.
A different approach is to carry out the purification on the MALDI probe surface itself, which avoids many sources of sample loss. For example, a small amount of powdered chromatographic packing placed on the MALDI target allows for the selective removal of interfering components (Rouse and Vath, 1996,
Anal Biochem.
238:82). Surface modified agarose beads have also been used for this purpose (Hutchens and Yip, 1993,
Rapid Commun. Mass Spectrom.
7:576). Furthermore, the use of films for sample supports for MALDI mass spectrometry of impure samples was patented by John S. Cottrell (U.S. Pat. No. 5,260,571), which discloses the application and use of thin films on the surface of the MALDI probe as a method of preparing a sample for analysis. An alternative technique consists of chemically modifying the probe surface by the addition of coatings such as nitrocellulose (Liu et al., 1995,
Anal. Chem.
67:3482) and Nafion (Bai et al, 1994,
Anal. Chem.
66:3423). As an extension of this approach, C-18 derivatized targets have been prepared (Brockman et al., 1997,
Anal. Chem.
69:4716). Basically, if the analyte of interest is selectively adsorbed onto the modified probe, interfering substances can be washed off, while the analyte is retained. However, in the aforementioned examples, modification of the probe surface for sample binding is time-consuming and the probes are good for only a limited number of uses. In addition, samples must still be transported to the MALDI-TOFMS laboratory by conventional means, that is, in solution and on ice.
Similarly, MALDI-TOFMS has been used recently to analyze hemoglobin from whole blood (Houston and Reilly, 1997,
Rapid Commun. Mass Spectrom.
11:1435). Rapid screening for hemoglobin abnormalities is of great importance as many health authorities now require the screening of new-borns' blood for hemoglobin-related diseases, as it has been shown that early detection of sickle cell disease significantly reduces infant mortality rates from this disease (Vichinsky et al., 1988,
Pediatrics
81:749). In the Houston and Reilly protocol (supra), whole blood samples are diluted and mixed with matrix solution. The resulting mixture is placed on a stainless steel MALDI probe and allowed to dry. While the MALDI spectra obtained with this method are of good quality, sample preparation requires a skilled mass spectrometrist familiar with MALDI matrix preparation methods. In addition, the analysis needs to be performed immediately or the sample processed and stored on ice.
The use of membranes as sample supports has recently been adopted as a means of both sample purification and sample delivery into the mass spectrometer (Vestling and Fenselau, 1995,
Mass Spectrom. Rev.
14:169; Strupat et al. in
Mass Spectrometry in the Biological Sciences
, A. L Burlingame and S. A. Carr editors, Humana Press: Totowa, N. J., 1996) p203). Several different membranes have been used for sample supports for MALDI mass spectrometry, two examples of which are: poly(vinylidene difluoride) (PVDF) (Vestling and Fenselau, 1994
Anal. Chem.
66:4371; Strupat et al., 1994,
Anal. Chem.
66:464) and polyether (Blackledge and Alexander, 1995,
Anal. Chem.
67:843). Specifically, these membranes have been used to prepare samples for MALDI-TOFMS from solution or following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). That is, purification of the analyte sample is performed prior to MALDI analysis. It is of note that deposition of aqueous protein solutions onto membrane supports has been shown to enhance MALDI signals for samples containing buffer components in higher concentrations than can generally be tolerated with traditional protocols. Subsequent purification by on-probe washing and/or enzymatic digestion results in low sample loss since proteins and peptides are bound fairly strongly to the membrane by ionic and hydrophobic interactions. However, the above-described membranes are porous and therefore have a heterogeneous surface which reduces the accuracy, sensitivity and resolution of the analysis. Specifically, the porosity permits distribution of the analyte and matrix within the membrane (Blackledge and Alexander, 1995,
Anal Chem.
67:843). This distribution of analyte within the porous surface reduces the amount of analyte available near the top of the membrane for sampling with the laser to bring about a MALDI-MS spectrum. Thus, increased laser irradiance is required to penetrate deep into the pores. This increased laser irradiance results in charging which causes an increase in the flight time, which significantly reduces spectral quality compared with metallic targets. The distribution also results in a non-uniform initial starting point for ions, again reducing the spectral quality by reducing the resolution.
Here we report the use of non-porous membranes as sample supports on MALDI probes. The membranes have a uniform surface, allowing for greater sensitivity and accuracy. Specifically, the non-porosity favours crystal growth on the surface of the membrane only, thereby providing enhanced spectral quality over membranes with porous structures. Studies were performed using polyurethane (PU) membranes as a

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