Focused Proteomics in Post-Mortem Human Spinal Cord

Ekegren, Titti; Hanrieder, Jörg; Aquilonius, Sten–Magnus; Bergquist, Jonas

doi:10.1021/pr060237f

Cited by 30 publications

(28 citation statements)

References 42 publications

(60 reference statements)

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“…Although mutations in dynein or its activator dynactin were demonstrated to lead to synapse disruption and neurodegeneration (14 -16), the effect of the mutations in slowing down dynein-mediated transport is not sufficient to create the harsh neurodegeneration observed in ALS (17,18), suggesting an additional mechanism. One possibility is a switch in the nature of the retrogradely transported cargo from survival signals to stress signals (19). Hence, a change in the composition of dynein complexes may underlie neurodegenerative and synapse elimination mechanisms.…”

Section: A315tmentioning

confidence: 99%

Proteomic Analysis of Dynein-Interacting Proteins in Amyotrophic Lateral Sclerosis Synaptosomes Reveals Alterations in the RNA-Binding Protein Staufen1

Gershoni-Emek

Mazza

Chein

et al. 2016

Molecular & Cellular Proteomics

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Synapse disruption takes place in many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the mechanistic understanding of this process is still limited. We set out to study a possible role for dynein in synapse integrity. Cytoplasmic dynein is a multisubunit intracellular molecule responsible for diverse cellular functions, including long-distance transport of vesicles, organelles, and signaling factors toward the cell center. A less well-characterized role dynein may play is the spatial clustering and anchoring of various factors including mRNAs in distinct cellular domains such as the neuronal synapse. Here, in order to gain insight into dynein functions in synapse integrity and disruption, we performed a screen for novel dynein interactors at the synapse. Dynein immunoprecipitation from synaptic fractions of the ALS model mSOD1 G93A and wild-type controls, followed by mass spectrometry analysis on synaptic fractions of the ALS model mSOD1 G93A and wild-type controls, was performed. Using advanced network analysis, we identified Staufen1, an RNA-binding protein required for the transport and localization of neuronal RNAs, as a major mediator of dynein interactions via its interaction with protein phosphatase 1-beta (PP1B). Both in vitro and in vivo validation assays demonstrate the interactions of Staufen1 and PP1B with dynein, and their colocalization with synaptic markers was altered as a result of two separate ALS-linked mutations: mSOD1 G93A and TDP43 A315T. Taken together, we suggest a model in which dynein's interaction with Staufen1 regulates mRNA localization along the axon and the synapses, and alterations in this process may correlate with synapse disruption and ALS toxicity. Molecular & Cellular

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Section: A315tmentioning

confidence: 99%

Proteomic Analysis of Dynein-Interacting Proteins in Amyotrophic Lateral Sclerosis Synaptosomes Reveals Alterations in the RNA-Binding Protein Staufen1

Gershoni-Emek

Mazza

Chein

et al. 2016

Molecular & Cellular Proteomics

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“…Material corresponding to approximately 450 µg of total protein for each whole spinal cord sample and 35 ng of total protein for microdissected motor neurons were then reduced, alcylated, enzymatically digested and desalted prior to ESI-FTICR-MS. Data were collected in Xmass  and the number of peptide clusters in each calibrated spectra calculated in MSwiz 0.3b, as previously described. 16 Peptide clusters were then compared to an in-house made database and proteins identified on a 95% significance level.…”

Section: Screening For Proteins In Whole Spinal Cord and Single Motormentioning

confidence: 99%

“…A highly suitable approach for this is, as previously described, laser-based microdissection that enables region-focused proteomics in an elegant way. 16,64 It has been estimated that about 10% of all proteomic studies already include tissue microdissection. 65 Since the application of microdissection techniques in proteomics has increased in the last 2 years, it is expected that more of these tissue-specific proteomic studies will be seen in the future.…”

Section: Clinical Samplesmentioning

confidence: 99%

“…Ramström et al 15 utilized the proteomic approach of on-line capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS), for advanced protein pattern classification and identification analyses in cerebrospinal fluid (CSF). Furthermore, by utilizing FTICR-MS in conjunction with nanoflow LC coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS) for sensitive proteomic profiling, Ekegren et al 16 identified proteins in minimal amounts of ALS postmortem spinal cord tissue. This study also includes an example of focused cell proteomics with the application of laser microdissection in combination with pressure catapulting (LMPC) for dissection of single motor neurons.…”

Section: Introductionmentioning

confidence: 99%

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Clinical perspectives of high‐resolution mass spectrometry–based proteomics in neuroscience—Exemplified in amyotrophic lateral sclerosis biomarker discovery research

2008

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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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“…In our laboratory we have been studying the neuroproteome since early 2000 developing and applying novel sample preparation and proteomic approaches in neurodegenerative diseases [7][8][9][10][11][12][13][14][15][16][17]. There are many other tools available for proteomic research including protein microarrays [18], proximity ligation assay [19], Luminex-based multiplex assay [20,21], Bio-Plex assay [22], etc.…”

Section: Introductionmentioning

confidence: 99%

Neuroproteomics tools in clinical practice

Shevchenko

Konzer

Musunuri

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) are characterized by neuronal impairment that leads to disease-specific changes in the neuronal proteins. The early diagnosis of these disorders is difficult, thus, the need for identifying, developing and using valid clinically applicable biomarkers that meet the criteria of precision, specificity and repeatability is very vital. The application of rapidly emerging technology such as mass spectrometry (MS) in proteomics has opened new avenues to accelerate biomarker discovery, both for diagnostic as well as for prognostic purposes. This review summarizes the most recent advances in the mass spectrometry-based neuroproteomics and analyses the current and future directions in the biomarker discovery for the neurodegenerative diseases.

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Focused Proteomics in Post-Mortem Human Spinal Cord

Cited by 30 publications

References 42 publications

Proteomic Analysis of Dynein-Interacting Proteins in Amyotrophic Lateral Sclerosis Synaptosomes Reveals Alterations in the RNA-Binding Protein Staufen1

Proteomic Analysis of Dynein-Interacting Proteins in Amyotrophic Lateral Sclerosis Synaptosomes Reveals Alterations in the RNA-Binding Protein Staufen1

Clinical perspectives of high‐resolution mass spectrometry–based proteomics in neuroscience—Exemplified in amyotrophic lateral sclerosis biomarker discovery research

Neuroproteomics tools in clinical practice

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