Tissue samples are then coated with organic compounds that act as matrix. Proper selection of matrix

is a critical step in MALDI to obtain good quality spectra. The most commonly used MALDI matrices are sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-hydroxybenzoic acid (DHB). SA is used for analyzing proteins with high molecular weight. CHCA and DHB are used for small molecules like peptides and lipids. The role of matrix in MALDI is to facilitate ablation and ionization of compounds in the sample. Uniform coating of the tissue section with matrix is important for efficient extraction and desorption of molecules from the tissue surface. Excessive matrix can cause migration of analytes in the tissue section. Conversely, insufficient or uneven deposition of matrix can check details lead to unstable and poor analyte signal. The most common techniques used for coating matrix are, pneumatic spraying [66], inkjet printing GSK2118436 [67], sublimation of matrix [68] and acoustic matrix deposition [69] because they produce a homogenous layer of small MALDI matrix crystals [70]. Several mass analyzers are used for IMS studies such as,

linear ion traps (LIT), orbitrap, QqTOF, and TOF/TOF instruments. TOF mass analyzers have no theoretical upper mass limit since TOF measures time required for an ion to travel from the ion source to the detector. Using this technique, It is possible to identify protein biomarker – for example, a 12,959 Da protein implicated in gentamicin-induced nephrotoxicity in rat was found to be transthyretin (Ser(28)-Gln(146)) [71]. For neuroscience study, a 2-D-IMS-visualization of MBP in mouse brain, including well defined corpus callosum region where MBP is highly localized. For neuroproteomic study, IMS was used to study 2-D visualization of protein expression in mouse brain structures [72]. Fig. 3 shows general workflow for MALDI imaging. Coronal sections of rat brain were analyzed to study the distribution of MBP. The image shows distribution of 14 kDa isoform of MBP in the rat

brain. It is also possible to combine brain IMS data with classic histology staining [73] or with MRI [74]. In terms of clinical translation, in principle, IMS can be applied to biopsy Farnesyltransferase and post-mortem brain tissue to examine protein localization or alteration. IMS analysis protocol has recently been derived for formalin-fixed paraffin-embedded tissue often obtained as clinical specimen [75]. With the different neuroproteomic techniques described here, one should select a fit-for-purpose method based on the requirements of the particular neuro-injury study and sample type available. Differential proteomics approach is best applied to neuro-tissue or cultured neural cell samples under two or more different experimental challenges. This approach is very useful during the discovery phase of protein changes, target or biomarker identification.