Protocol for the Bottom-Up Proteomic Analysis of Mouse Spleen

Abstract

Summary This protocol describes the comparative proteomic profiling of the spleen of wild type versus mdx-4cv mouse, a model of dystrophinopathy. We detail sample preparation for bottom-up proteomic mass spectrometry experiments, including homogenization of tissue, protein concentration measurements, protein digestion, and removal of interfering chemicals. We then describe the steps for mass spectrometric analysis and bioinformatic evaluation. For complete details on the use and execution of this protocol, please refer to Dowling et al. (2020 ).

Urine proteomics as a non-invasive approach to monitor exertional rhabdomyolysis during military training

“…FABP3 had a higher association with muscle weakness in polymyositis and dermatomyositis patients than CK and myoglobin serum levels (Zhang et al, 2016). A recent study showed that serum levels of FABP3 might represent a novel biomarker for Duchenne muscular dystrophy (Dowling et al, 2020). To the best of our knowledge, this is the first report that shows an increased abundance of FABP3 in human urine after strenuous exercise and its correlation with serum biomarkers of muscle damage.…”

Mass Spectrometric Profiling of Extraocular Muscle and Proteomic Adaptations in the mdx-4cv Model of Duchenne Muscular Dystrophy

“…Verification analyses were carried out with samples derived from a minimum of 4 wild type and 4 dystrophic mice. Comparative tissue proteomics was carried out by standardized procedures, as previously described in detail [ 40 , 41 ]. Mice were sacrificed in the Bioresource Unit of the University of Bonn and muscle specimens were quick-frozen in liquid nitrogen and then transported on dry ice to Maynooth University [ 42 ].…”

“…The label-free liquid chromatography mass spectrometric analysis of EOMs from wild type versus mdx-4cv mice was carried out using a Thermo Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Details of the proteomic workflow describing all preparative steps and analytical procedures using data-dependent acquisition, as well as bioinformatic data handling, were recently outlined in detail [ 41 ]. A Thermo UltiMate 3000 nano system was used for reverse-phased capillary high-pressure liquid chromatography and directly coupled in-line with the Thermo Orbitrap Fusion Tribrid mass spectrometer.…”

“…The Progenesis QI programme calculated the mean abundance for individual protein species in each experimental condition to determine the maximum fold change for particular proteins. Condition-vs-condition matrixes with mean values were then used to determine the maximum fold change between any two condition's mean protein abundances [41]. The raw MS files generated by this proteomic study were deposited under the unique identifier 'j4867' to the Open Science Foundation (https://osf.io/j4867/ (accessed on 15 June 2021)).…”

Mass Spectrometry-Based Proteomic Technology and Its Application to Study Skeletal Muscle Cell Biology

“…For bottom-up proteomics, a variety of sample preparation approaches have been developed for optimum protein extraction [150][151][152], including the widely employed filteraided sample preparation (FASP) method that can be used to exchange buffers and remove incompatible detergents prior to MS-based analysis [153]. The routine bottom-up proteomic analysis pipeline can be divided into a series of consecutive steps [152] comprising (i) sample harvesting from complex cellular mixtures, (ii) sample pre-treatment, such as enrichment procedures using differential centrifugation or affinity isolation of protein constellations, or depletion procedures to enrich low-abundance proteins, (iii) initial sample protection from excess proteolysis using suitable protease inhibitor cocktails, (iv) efficient homogenization using mechanical grinding approaches or sample pulverization with liquid nitrogen, (v) protein extraction via precipitation or other biochemical methods, (vi) protein quantification using sensitive dye binding or other reliable assay systems, (vii) chemical reduction and alkylation of protein samples, (viii) protein digestion using enzymes such as trypsin for the generation of distinct peptide populations, (ix) peptide fractionation and MS analysis, (x) protein identification, and (xi) systems bioinformatic analysis of identified proteoforms and their PTMs [154][155][156]. Figure 2 compares the main differences in the preparation of protein samples between top-down proteomics and bottom-up proteomics, i.e., the isolation of intact proteoforms versus the enrichment of protein fractions.…”