Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressing disease of the central nervous system that is characterized by motor neuron degeneration in the brainstem and the spinal cord. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry is an emerging powerful technique that allows for spatially resolved, comprehensive, and specific characterization of molecular species in situ. In this study, we report for the first time the MALDI imagingbased spatial protein profiling and relative quantification of post-mortem human spinal cord samples obtained from ALS patients and controls. In normal spinal cord, protein distribution patterns were well in line with histological features. For example, thymosin beta 4, ubiquitin, histone proteins, acylCoA-binding protein, and macrophage inhibitory factor were predominantly localized to the gray matter. Furthermore, unsupervised statistics revealed a significant reduction of two protein species in ALS gray matter. One of these proteins (m/z 8451) corresponds to an endogenous truncated form of ubiquitin (Ubc 1-76), with both C-terminal glycine residues removed (Ubc-T/Ubc 1-74). This region-specific ubiquitin processing suggests a disease-related change in protease activity. These results highlight the importance of MALDI mass spectrometry as a versatile approach to elucidate molecular mechanisms of neurodegenerative diseases. Keywords: amyotrophic lateral sclerosis, imaging mass spectrometry, matrix-assisted laser desorption/ionization, post-mortem spinal cord, tissue imaging. J. Neurochem. (2013) 124, 695-707. As the worldwide proportion of elderly people above 60 is increasing constantly, the prevalence for age-related diseases is on the rise, particularly with respect to emerging newly industrialized countries (W.H.O 2002). These diseases of affluent societies include cardiovascular diseases, chronic obstructive pulmonary disease, cancer, diabetes and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis (ALS) (Hirtz et al. 2007). ALS, also referred to as Lou Gehring's Disease or Maladie de Charcot, is characterized by irreversible degeneration of motor neurons in the spinal cord, brainstem, and cortex. This results in increasing muscle weakness and muscle atrophy. Most commonly, people between age 40 and 60 are affected and worldwide prevalence of ALS has been reported to be 2-4 cases per 100 000 a year (Hirtz et al. 2007). ALS is a very progressive, fatal disorder, and patients have an average life expectancy of 3-5 years after diagnosis. Most of ALS cases are idiopathic in nature; however, 10% have been Clement et al. 2003;Cristini 2006). The limited knowledge of the disease results in further drawbacks such as the inaccuracy and time delay for diagnosis. This is because particularly early disease stages of ALS are shared by other neurological disorders, including multifocal motoric neuropathy (www.alsa.org). As the major biomolecular mechanisms...

With a highly sensitive electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) system, proteins were identified in minimal amounts of spinal cord from patients with the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and compared to proteins in spinal cord from control subjects. The results show 18 versus 16 significantly identified (p < 0.05) proteins, respectively, all known to be found in the central nervous system. The most abundant protein in both groups was the glial fibrillary acidic protein, GFAP. Other proteins were, for example, hemoglobin alpha- and beta chain, myelin basic protein, thioredoxin, alpha enolase, and choline acetyltransferase. This study also includes the technique of laser microdissection in combination with pressure catapulting (LMPC) for the dissection of samples and specific neurons. Furthermore, complementary experiments with nanoLC-matrix assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS) confirmed the results of the ESI-FTICR MS screening and provided additional results of further identified proteins.

Biomarker discovery is a central application in today's proteomic research. There is an urgent need for valid biomarkers to improve diagnostic tools and treatment in many disorders, such as the rapidly progressing neurodegenerative disorder amyotrophic lateral sclerosis (ALS) that has a fatal outcome in about 3 years and yet no curative treatment. Screening for clinically relevant biomarkers puts high demands on high-throughput, rapid and precise proteomic techniques. There is a large variety in the methods of choice involving mainly gel-based approaches as well as chromatographic techniques for multi-dimensional protein and peptide separations followed by mass spectrometry (MS) analysis. This special feature article will discuss some important aspects of MS-based clinical proteomics and biomarker discovery in the field of neurodegenerative diseases and ALS research respectively, with the aim to provide a prospective view on current and future research aspects in the field. Furthermore, examples for application of high-resolution MS-based proteomic strategies for ALS biomarker discovery will be demonstrated with two studies previously reported by our group. These studies include among others, utilization of capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) for advanced protein pattern classification in cerebrospinal fluid (CSF) samples of ALS patients as well as highly sensitive protein identification in minimal amounts of postmortem spinal cord tissue and laser micro-dissected motor neurons using FT-ICR-MS in conjunction with nanoflow LC coupled to matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (LC-MALDI-TOF-TOF-MS).

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Objectives‐ To investigate if degeneration of motor neurons in amyotrophic lateral sclerosis (ALS) is related to altered levels of the apoptosis regulating proteins Bcl‐2 and Bax. In addition, immuno‐reactivity of the cysteine protease ICH‐1L and detection of motor neurons with DNA fragmentation, indicative of apoptosis, was also studied. Material and methods‐ The immunoreactivity of Bcl‐2, Bax and ICH‐1L were compared in ALS and control spinal cord motor neurons by immunohistochemical analysis and motor neurons with DNA fragmentation were identified by the TUNEL‐method. Results‐ The results demonstrate an increased expression of Bax in the ALS material as compared to controls but no change in Bcl‐2 and ICH‐1L expressions. Moreover, a larger proportion of motor neurons stained positive for TUNEL in ALS spinal cords. Conclusion‐ Present study suggest an upregulation of the cell death promoting protein Bax and increased DNA degradation, indicative of apoptosis, in spinal motor neurons of ALS patients.

The objective of this study was to develop and apply a novel multiplex panel of solid-phase proximity ligation assays (SP-PLA) requiring only 20 μL of samples, as a tool for discovering protein biomarkers for neurological disease and treatment thereof in cerebrospinal fluid (CSF). We applied the SP-PLA to samples from two sets of patients with poorly understood nervous system pathologies amyotrophic lateral sclerosis (ALS) and neuropathic pain, where patients were treated with spinal cord stimulation (SCS). Forty-seven inflammatory and neurotrophic proteins were measured in samples from 20 ALS patients and 15 neuropathic pain patients, and compared to normal concentrations in CSF from control individuals. Nineteen of the 47 proteins were detectable in more than 95% of the 72 controls. None of the 21 proteins detectable in CSF from neuropathic pain patients were significantly altered by SCS. The levels of the three proteins, follistatin, interleukin-1 alpha, and kallikrein-5 were all significantly reduced in the ALS group compared to age-matched controls. These results demonstrate the utility of purpose designed multiplex SP-PLA panels in CSF biomarker research for understanding neuropathological and neurotherapeutic mechanisms. The protein changes found in the CSF of ALS patients may be of diagnostic interest.

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