To determine region-specific differences in protein intensities that are ALS-related, unpaired statistical analysis of averaged peak bin integration data of the respective ROI (GM, VH, DH, and WM) of the ALS and control samples was performed. The number of spectra varied for each ROI in each sample, respectively. In addition to the number of GM and WM spectra that is described above, the different regions of the gray matter comprised 39–125 spectra for the ventral horn and 23–68 spectra for the dorsal horn region of the GM. Unpaired statistical analysis of the different regions of interest (GM, VH, DH, and WM) in between the two sample groups showed significant changes in the gray matter; however, no consistent change for white matter proteins was observed. Therefore, histology directed experiments that were performed in duplicates for each sample were focused on the gray matter region (GM) and its subregions (DH, VH). The number of acquired spectra for each respective ROI was similar to the imaging data. Here, a number of 82–148 spectra per patient were collected for the GM, 29–42 for the dorsal horn, and 47–106 for the ventral horn, respectively. Although no consistent differences were observed for the subregions (DH, VH), for two protein peaks including m/z 8429 and m/z 8451, a consistently significant lower peak intensity (p < 0.05) was observed in the whole gray matter (GM) of ALS patients compared with the whole GM of controls in all three replicate experiments (Fig. 4). Interestingly, none of the proteins reported previously to be changed in ALS were found changed in the present study (Ekegren et al. 2006). However, this might be because of the different experimental strategies employed, as the present study was performed on proteins with a molecular weight below 20–30 kDa (corresponding to the MALDI mass range) as a result of mass cut filtration or selective gel range excision.
The data analysis results were followed up a series of validation experiments aiming to determine the correct identity of these two protein species. First, the spatial ion intensity distributions were investigated closely to clarify whether m/z 8451 is solely a sodium adduct peak of 8429 (mass shift from protonated peak to sodium adduct peak: 22 Da) which in turn should result in identical localization. However, the ion distribution patterns were found to be different suggesting that both peaks originate from different protein species (Fig. 4a). Another aspect of this is that cation adduct peaks (M+Na+, M+K+) are typically significantly smaller than the corresponding main analyte peak. Furthermore, we performed C8 reversed phase HPLC of intact proteins extracted from spinal cord followed by fraction collection. The individual fractions were spotted onto a MALDI target plate for protein LC-MALDI analysis allowing for targeted follow-up analysis of intact protein peaks of interest. Again, assuming the two protein peaks were originating from the same protein and are a sole cationic adduct observed in MS would result in identical peak retention during liquid chromatography. Here, the two protein peaks were not found to exhibit a similar retention behavior further strengthening the hypothesis previously deduced from the ion images that these two protein peaks originate from different protein species. For protein identification, the corresponding fractions containing the two protein peaks of interest were subjected to tryptic digestion followed by LC-MSMS analysis of the enzymatic cleavage products. Detection and identification of the protein peak m/z 8429 have not been reported previously, and the here-reported identification experiments using bottom-up proteomics did not yield any results for this particular mass peak. The mass peak m/z 8451, however, has been previously reported to correspond to C-terminally truncated ubiquitin (Meistermann et al. 2006; Hardesty et al. 2011). Indeed, database analysis of the LC-MSMS data of the respective protein fraction showed identification of ubiquitin as one of the major protein matches. The other hits comprised myelin basic protein and ACBP. This is well in line with other major peaks observed in the MALDI data of the respective HPLC fraction including m/z 18418 and m/z 9964 previously assigned to myelin basic protein and ACBP, respectively (Table 2). Interestingly, Ubc-T is not an artifact due to unspecific cleavage, as its intensity distribution pattern differs significantly from intact ubiquitin, and points rather to a disease-related processing pattern of ubiquitin in human spinal cord (Fig. 2, panel b). Ubc-T has previously reported as potential biomarker of breast cancer as revealed by surface enhanced laser desorption/ionization mass spectrometry (Goncalves et al. 2008). During Ubc-mediated proteasomal protein degradation, C-terminal removal of two glycines leads to inactivation of Ubc. However, differential regulation of Ubc-T in contrast to steady-state Ubc levels as reported by Goncalves et al. points toward a distinct biological function of Ubc-T and Ubc processing, respectively, that evidently require further investigation (Goncalves et al. 2008). Previous studies have demonstrated that the bioconversion of ubiquitin 1–76 to ubiquitin 1–74 (Ubc-T) is predominantly mediated by cathepsin B, a lysosomal cysteine protease and member of the peptidase 1 family (Herring 2009). Interestingly, microarray-based gene expression studies in human ALS, as well as on experimental ALS in mice, have identified significant changes of cathepsin B mRNA levels in spinal cord tissue, however with contradicting results (Kikuchi et al. 2003; Offen et al. 2009). For example, a decrease of Ubc-T could consequently point toward reduced cathepsin B activity, which is supported by data on post-mortem human spinal cord reported by Kikuchi et al. 2003). where decreased levels of cathepsin B mRNA were detected in ALS patients compared with controls (Kikuchi et al. 2003). Alternatively, decreased Ubc-T levels can also be a result of decreased Ubc levels and unspecific enzymatic cleavage. Decreased ubiquitin levels can be directly related to formation of Ubc-positive protein inclusions. Here, recruited Ubc is incorporated in the protein aggregates by forming stable covalent bonds, which in turn decreases the level of free Ubc molecules that can be detected by MALDI mass spectrometry. Either way, ubiquitin regulation and activity seem to be significantly affected in ALS. As second means of validation of MALDI-IMS results, double-antigen immunohistochemistry for the neuronal nuclear antigen NeuN (Fig. 5c, arrows) and ubiquitin (Ubc) confirmed neuronal loss and a decrease in Ubc immunoreactivity in the gray matter of ALS patients (n = 3 in each group). NeuN was chosen to monitor neuronal loss in ALS as well as target neuronal ubiquitin concentration specifically. However, the Ubc antibody recognizes both free and conjugated ubiquitin. In addition, the immunogen was the full-length human ubiquitin, which raises the possibility that this antibody also recognizes the short (-GlyGly, −114 Da) form of recycled ubiquitin. Lipofuscin autofluorescence was abundant in all sections and is seen as yellow granular staining, sometimes surrounding the nucleus. Due to these interferences, a quantitative statement was impossible to deduce from the immunohistochemistry results. We performed western blot experiments on gray matter protein extracts of all patient samples to quantify the degree of changes in abundance of ubiquitin species. The results show indeed that ubiquitin species are decreased by 57–53% in ALS gray matter samples compared with controls (Fig. 5 a, b; p < 0.05; Figure S2). These results can be regarded as further confirmation that there is an overall decrease of Ubc-related species in ALS as observed in MALDI-IMS, probably related to impaired protein turnover. However, due to lack of antibody specificity, differentiation between the intact and truncated Ubc is not possible. In addition, the 114 Da difference between Ubc and Ubc-T was undetectable by western immunoblotting. This makes it difficult to draw conclusions whether or not C-terminal Ubc processing is related to ALS, although the MALDI results show that Ubc-T but not Ubc peak values were reduced in ALS, further highlighting the superiority in terms of molecular specificity of this technology. Selective decrease in Ubc-T but not Ubc would support the theory that decreased cathepsin B activity is involved in ALS pathology (Fig. 4) as previously suggested (Kikuchi et al. 2003). However, one has to keep in mind that MALDI MS as a semi-quantitative technique is characterized by a certain variance due to its susceptibility to matrix effects, which could explain why intact Ubc 1–76 was not found consistently decreased in ALS. These include, for example, ion suppression as a result of impurities and changes in relative analyte concentration This is significantly aggravated when patient material is used where large individual variations are inherent as well as other factors including post-mortem time, sample retrieval, sample age and sample storage contribute to biological sample variation significantly. As shown in the present study, only a very small number of protein species was identified that showed consistent and significant changes in peak intensity in all replicate experiments. Hence, these results perhaps detect only the protein peaks that exhibited the most consistent changes caused by ALS and might thus represent only an excerpt of all changed protein species including ubiquitin 1-76. This also highlights the need for sufficient sample size and well-controlled experimental parameters for sample handling to minimize biological variation. For the present study, these factors are compromised due to the fact that human post-mortem material is used and sample access is limited due to the low prevalence of ALS. However, the here-presented results demonstrate that profound optimization of sample preparation and data acquisition parameters for minimizing technical variation allows for MALDI-IMS-based investigation of such diverse biological samples with reasonable and reproducible outcome. In summary, the observed results suggest that regulation of ubiquitin and regional ubiquitin metabolism is potentially connected to pathophysiological mechanisms underlying ALS. These data support previous observations that impaired protein turnover is a hallmark in ALS pathology.