Comparison between vacuum sublimed matrices and conventional dried droplet preparation in MALDI-TOF mass spectrometry

Thorsten W. Jaskolla 0 1 4 Michael Karas 0 1 4 Udo Roth 0 1 2 Kerstin Steinert 0 1 2 Christoph Menzel 0 1 2 Karsten Reihs 0 1 3 0 Address reprint requests to Graduate engineer T. Jaskolla, Institute of Pharmaceutical Chemistry , Max-von-Laue-Str. 9, Goethe University Frank- furt , D-60438 Frankfurt, Germany 1 Published online February 12, 2009 Received October 30, 2008 Revised January 28, 2009 Accepted February 4, 2009 2 Qiagen GmbH, Hilden, Germany 3 AMF GmbH, Kln, Germany 4 Cluster of Excellence Macromolecular Complexes, Institute of Pharmaceutical Chemistry, Goethe University , Frankfurt, Germany The properties of several cinnamic acid compounds used as matrices for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were investigated as standard dried droplet (DD) and vacuum sublimed preparations. The differences between both preparation methods were analyzed with regard to matrix grain size, internal ion energy, initial velocity, analyte intensity, and analyte incorporation depth. Some of the used cinnamic acid derivatives exhibit clearly reduced grain sizes as sublimed preparations compared with standard DD approaches. In these cases higher effective temperatures could be measured accompanied by increased analyte intensities, which can be explained by stronger volatilization processes caused by a hindered heat dissipation resulting in a raised analyte transfer into the gas phase. For all sublimed compounds, a strong increase of the initial ion velocity compared with DD preparations could be measured. Higher initial ion velocities correlate with a decrease in internal ion energy which might be attributed to the very uniform crystal morphology exhibited by sublimed compounds. For sublimed matrices without reduced grain size, at least slightly higher analyte intensities could be detected at raised laser fluences. Analyte accumulation in the uppermost matrix layers or the detected higher ion stability can be explanations for these results. (J Am Soc Mass Spectrom 2009, 20, 1104 -1114) 2009 American Society for Mass Spectrometry - erties compared with standard dried droplet (DD) preparations: The main advantage of sublimed matrix spots compared with standard DD preparations are higher analyte sensitivities [14 17]. This is likely related to the formation of considerably smaller crystals and narrow size distribution of thin-film sublimed matrices, such as -cyano-4-hydroxycinnamic acid (CHCA), compared with DD preparations with comparatively large particle sizes and broader size distribution [15]. As a consequence of this, the specific matrix surface area as well as the amount of potential binding places increases compared with standard DD precipitates, which will result in increased analyte binding properties. Additionally, the process of matrix preparation and spotting is omitted, resulting in reduced sample preparation work, which seems beneficial especially for highthroughput analysis. Moreover, the sublimation process purifies the matrix compound leading to matrix layers of improved purity that homogeneously cover the entire sample spot area. It also has been shown that small and evenly distributed crystals exhibit higher spot-tospot reproducibility compared with standard DD preparations [18], with only partial crystal coverage of the spot surface necessitating the selection of individual crystals or random walking over the spot. Therefore, Mspectrometry (MALDI-MS) [13] has evolved atrix-assisted laser desorption ionization mass into an essential tool for chemical analysis of biomolecules. Ever increasing numbers of samples call for automation in high-throughput workflows. One of the key areas for progress in automated MALDI analyses consists of improved sample preparation techniques. Numerous efforts have been conducted for improving the preparation step, e.g., the generation of thin uniform matrix layers with high shot-to-shot reproducibility using the aerospray technique by combination of matrix and nitrocellulose [4] or for analysis of synthetic polymers [5]. Also, electrospray deposition and vacuum deposition were reported to yield homogeneous sample preparations with increased analyte intensities [6 9]. Matrix deposition by sublimation for MALDI imaging has been reported to yield more intense signals with less alkali adducts compared with the electrospray technique [10]. Additionally, sublimed matrix spots deposited on extremely hydrophobic surfaces described in [1114] offer a variety of beneficial propadditional time and laser shots are required due to analysis of noncovered or partially covered spot areas. In combination with other features like precisely defined spot size, shape, and location, this approach enables more homogeneous sample preparation conditions and higher reproducibility [14]. Nevertheless, the influence of sublimation on matrix properties is not yet fully understood. Therefore, physicochemical parameters that seem important for the ablation process are compared in detail in this work for standard DD and sublimed matrix preparations for the most widely used matrix CHCA and structurally similar derivatives, which are suitable matrix compounds. Materials and Reagents All chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany) and were of analytical grade, except p-tolualdehyde, sinapinic acid (SA), and trifluoroacetic acid (TFA), which were of the highest grade available. The in-solution bovine serum albumin (BSA) digest (Starter Kit for MALDI-TOF MS, Part-No.: 208241, Lot: 2004-208241-001) and CHCA (Part-No.: 201344, Lot: 2007-201344-001) were purchased from Bruker Daltonics (Bremen, Germany). Vanillin (99%) was obtained from Acros (Nidderau, Germany). Acetonitrile (ACN) and methanol (MeOH) were both Rotisolv HPLC gradient grade and purchased from Roth (Karlsruhe, Germany). Water was purified by an ion-exchange system (Milli-Q, Millipore (Schwalbach, Germany). The Sequazyme Mass Standards Kit (calibration mixture 1 2) was obtained from Applied Biosystems (Darmstadt, Germany). Quaternary p-methoxybenzylpyridinium chloride was synthesized by the corresponding benzyl chloride and pyridine as described previously [19]. GELoader Tips were ordered from Eppendorf (Wesseling-Berzdorf, Germany). Sample holders (Mass Spec Turbo Chip Holders) were courtesies from Qiagen N.V. (Hilden, Germany). Synthesis of CHCA Derivatives -Cyano-4-methylcinnamic acid (4-Me-CCA), -cyano4-hydroxy-3-methylcinnamic acid (4-HO-3-Me-CCA), and -cyano-4-hydroxy-3-methoxycinnamic acid (4HO-3-MeO-CCA) were synthesized by standard Knoevenagel condensation reactions using cyanoacetic acid and substituted benzaldehydes. Ammonium acetate was used as catalyst. In a typical approach, 2 g cyanoacetic acid (1 eq), 0.9 eq of the benzaldehyde and 0.15 eq of ammonium acetate were refluxed with stirring in toluene. After quantitative separation of the byproduct water by a water separator the reaction mixture was cooled and filtere (...truncated)