A review of complementary separation methods and matrix assisted laser desorption ionization-mass spectrometry imaging: Lowering ଝ sample complexity a,b,c a,b,∗ Karolina Skrᡠskovᡠ, Ron M.A. Heeren a Biomolecular Imaging Mass Spectrometry (BIMS) Group, FOM-Institute AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands b TI-COAST, Science Park 904, 1098 XH Amsterdam, The Netherlands c Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic a b s t r a c t Keywords: MALDI mass spectrometry imaging Ionization suppression Liquid chromatography Gel electrophoresis Blotting Polymeric membranes Contents 1. Introduction . 2 2. Separations for effective sample clean-up. 3 2.1. Sample supports for MALDI-MS . 3 2.1.1. Polymeric membranes . 3 2.1.2. MALDI sample supports with a modified surface . 3 2.2. Washing protocols for MALDI-MSI of tissue samples. 4 3. Separations within defined spatial dimensions . 4 3.1. Planar layout: one- and two-dimensional separations . 4 3.1.1. Gel electrophoresis . 5 3.1.2. Thin layer chromatography . 6 3.2. Blotting: adding the z-dimension . 7 3.2.1. Blotting TLC plates . 7 3.2.2. Blotting gels. 8 3.2.3. Blotting tissue sections . 9 4. One step blotting and digestion . 9 4.1. Protein identification strategies for MS . 9 4.2. Molecular scanner . 9 ଝ Presented at the 39th International Symposium on High-Performance Liquid-Phase Separations and Related Techniques, Amsterdam, Netherlands, 16–20 June 2013. ∗ Corresponding author at: FOM-Institute AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands. Tel.: +31 20 7547100; fax: +31 20 7547290. E-mail address: [email protected] (R.M.A. Heeren). 5. Column and capillary separations . 11 5.1. Direct MALDI interface . 11 5.2. Validation and corroboration of MSI results . 11 6. Conclusions and future perspectives . 11 Acknowledgements . 12 References . 12 1. Introduction composition of tissue sections. The term MALDI mass spectrometry imaging (MSI) has officially been used since that time, although its Matrix-assisted laser desorption ionization (MALDI) introduced basic principle was already known and applied for MS analysis of, in Karas et al. [1] pushed the borders of laser desorption ion- e.g. gels and TLC plates. MALDI-MSI was a logical junction between ization (LDI) techniques forward by enabling ionization of large the MALDI technique that emerged in the late 1980s and secondary (bio)polymers. Ultimately the ability to analyze intact macro- ion mass spectrometry (SIMS) imaging that was already well estab- molecules led to the emergence of new analytical disciplines such lished at the time [9]. During a MALDI-MSI experiment a sample as proteomics and metabolomics. The groundbreaking improve- surface is probed with a laser beam in a predefined raster form and ment was based on the use of matrix. Typically a small organic a mass spectrum is recorded at each position. As a result, position compound, the matrix has a triple impact on the desorption- correlated mass spectra are collected. The final image is defined by ionization process. (i) It separates individual molecules of the the x,y coordinates within a Cartesian grid, and the intensities of analyte from each other, thus minimizing analyte aggregation. (ii) a particular m/z visualized in a different colour range [10–12]. An Matrix molecules absorb the initial energy of a laser pulse. As a con- image can be constructed for every single m/z present in the spectra. ␮ sequence, the molecules of analyte do not undergo direct photon Common samples analyzed with MALDI-MSI are thin (10–20 m) induced fragmentation and organic compounds up to several tens tissue sections [13], but in principle any sample thin and flat enough to hundreds of kilo Daltons can be analyzed. (iii) It promotes ion- can be explored by MALDI-MSI. molecule reactions in the gas phase (MALDI plume), that results in In general, MSI offers several advantages over the other imag- formation of pseudomolecular ions from analyte molecules [2–4]. ing technologies such as positron emission tomography, computer The most common sample preparation for a MALDI experi- assisted X-ray tomography, nuclear magnetic resonance imaging, ment is the dry droplet method: a solution of analyte(s) is mixed quantitative whole body autoradiography (QWBA), or immunohis- with a matrix solution and small droplets of the mixture are spot- tochemistry (IHC). First of all, MSI does not require any knowledge ted onto a metal target. Upon the droplet drying, co-crystals of on the targeted analytes and has no need for any labels. It is a true matrix and analyte are formed. The actual desorption-ionization discovery tool. Furthermore, it allows for highly precise and specific process is initiated by exposure to one or more laser pulses. Nowa- identification of many hundreds of compounds next to each other days UV lasers such as nitrogen (337 nm) or Nd:YAG (355 nm) while preserving their spatial distribution [12,13]. Indeed, the spa- are mostly employed, and also IR-MALDI with lasers emitting at tial information is one of the most significant contributions of MSI. 2.94 ␮m are used in analytical practice [5]. The laser shots cause Nevertheless, MALDI-MSI is affected by the general weak points of desorption of the matrix-analyte cluster ions as well as matrix and MALDI described above, thus suffering from several limitations. analyte neutrals from the laser interaction region. The analyte ions The simplest MALDI-MSI sample preparation protocol is based emerge after ion-molecule reactions with the matrix ions (ideally on cutting a sample (e.g. a mouse brain) into thin sections and cov- after gas-phase protonation) or cluster desolvation in the MALDI ering them with a layer of matrix [13]. The overlap problem of plume [6]. For its pulsed character, MALDI is preferably coupled to matrix and its cluster peaks is manifested for the molecules within time-of-flight (TOF) mass analyzers. the matching mass range. Moreover, as the laser beam rasters over On one hand, the employment of matrix enabled analysis of the sample, all molecules present on the sample surface can theo- high molecular mass species, on the other, matrix presence ham- retically be desorbed and ionized. Some molecules tend to ionize pers analysis of the compounds at the low end of the mass range. more easily, especially when they are present in a higher amount, Because matrix is typically a compound with the molecular mass hence they contribute to ionization suppression effects. These below 500 Da (e.g. sinapinic acid, 2,5-dihydroxybenzoic acid, ␣- can, together with a limited dynamic range of MALDI-TOF mass cyano-4-hydroxycinnamic acid (CHCA), etc.) the analysis of low spectrometers, lead to incomplete data since the low-abundant molecular mass species (<500 Da) is complicated by the overlap- molecular species remain “hidden”. Lowering.