laser-microdissection-based-protocol-for-lc-msms-analysis-proteomic

Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. ...

Ruhr-University Bochum

Protein sequence databases play a crucial role in the majority of the currently applied mass-spectrometry-based proteomics workflows. Here UniProtKB serves as one of the major sources, as it combines the information of several smaller databases and enriches the entries with additional biological information. For the identification of peptides in a sample by tandem mass spectra, as generated by data-dependent acquisition, protein sequence databases provide the basis for most spectrum identification search engines. In addition, for targeted proteomics approaches like selected reaction monitoring (SRM) and parallel reaction monitoring (PRM), knowledge of the peptide sequences, their masses, and whether they are unique for a protein is essential. Because most bottom-up proteomics approaches use trypsin to cleave the proteins in a sample, the tryptic peptides contained in a protein database are of great interest. We present a database, called MaCPepDB (mass-centric peptide database), that consists of the complete tryptic digest of the Swiss-Prot and TrEMBL parts of UniProtKB. This database is especially designed to not only allow queries of peptide sequences and return the respective information about connected proteins and thus whether a peptide is unique but also allow queries of specific masses of peptides or precursors of MS/MS spectra. Furthermore, posttranslational modifications can be considered in a query as well as different mass deviations for posttranslational modifications. Hence the database can be used by a sequence query not only to, for example, check in which proteins of the UniProt database a tryptic peptide can be found but also to find possibly interfering peptides in PRM/SRM experiments using the mass query. The complete database contains currently 5 939 244 990 peptides from 185 561 610 proteins (UniProt version 2020_03), for which a single query usually takes less than 1 s. For easy exploration of the data, a web interface was developed. A REST application programming interface (API) for programmatic and workflow access is also available at https://macpepdb.mpc.rub.de.

MaCPepDB: A Database to Quickly Access All Tryptic Peptides of the UniProtKB.

Cerebrospinal fluid (CSF) diagnostics has emerged as a valid tool for a variety of neurological diseases. However, CSF diagnostics has been playing a subordinate role in the diagnosis of many neurological conditions. Thus, in the multitude of neuromuscular diseases in which motor neurons are affected, a CSF sample is rarely taken routinely. However, CSF diagnostics has the potential to specify the diagnosis and monitor the treatment of neuromuscular disorders. In this review, we therefore focused on a variety of neuromuscular diseases, among them amyotrophic lateral sclerosis (ALS), peripheral neuropathies, and spinal muscular atrophy (SMA), for which CSF diagnostics has emerged as a promising option for determining the disease itself and its progression. We focus on potentially valuable biomarkers among different disorders, such as neurofilaments, cytokines, other proteins, and lipids to determine their suitability, differentiating between different neurological disorders and their potential to determine early disease onset, disease progression, and treatment outcome. We further recommend novel approaches, e.g., the use of mass spectrometry as a promising alternative techniques to standard ELISA assays, potentially enhancing biomarker significance in clinical applications.

Maximilian Wulf

Medizinisches Proteom-Center,
Medical Faculty,
Medical Proteome Analysis,
Center for Proteindiagnostics (PRODI),
Medizinisches Proteom-Center, Medical Faculty,
Medical Proteome Analysis, Center for Proteindiagnostics (PRODI)

Ruhr-University Bochum

Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

Maximilian Wulf

Medizinisches Proteom-Center,Medical Faculty,Medical Proteome Analysis,Center for Proteindiagnostics (PRODI),Medizinisches Proteom-Center, Medical Faculty,Medical Proteome Analysis, Center for Proteindiagnostics (PRODI)

Ruhr-University Bochum

Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

Medizinisches Proteom-Center,
Medical Faculty,
Medical Proteome Analysis,
Center for Proteindiagnostics (PRODI),
Medizinisches Proteom-Center, Medical Faculty,
Medical Proteome Analysis, Center for Proteindiagnostics (PRODI)