An update on multiplexed mass spectrometry-based lysosomal storage disease diagnosis.

Lysosomal storage disorders (LSDs) are a type of inherited metabolic disorders in which biomolecules, accumulate as a specific substrate in lysosomes due to specific individual enzyme deficiencies. Despite the fact that LSDs are incurable, various approaches, including enzyme replacement therapy, hematopoietic stem cell transplantation, or gene therapy are now available. Therefore, a timely diagnosis is a critical initial step in patient treatment. The-state-of-the-art in LSD diagnostic uses, in the first stage, enzymatic activity determination by fluorimetry or by mass spectrometry (MS) with the aid of dry blood spots, based on different enzymatic substrate structures. Due to its sensitivity, high precision, and ability to screen for an unprecedented number of diseases in a single assay, multiplexed tandem MS-based enzyme activity assays for the screening of LSDs in newborns have recently received a lot of attention. Here, (i) we review the current approaches used for simultaneous enzymatic activity determination of LSDs in dried blood spots using multiplex-LC-MS/MS; (ii) we explore the need for designing novel enzymatic substrates that generate different enzymatic products with distinct molecular masses in multiplexed-MS studies; and (iii) we give examples of the relevance of affinity-MS technique as a basis for reversing undesirable immune-reactivity in enzyme replacement therapy.

Applications of MALDI-MS/MS-Based Proteomics in Biomedical Research.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is one of the most widely used techniques in proteomics to achieve structural identification and characterization of proteins and peptides, including their variety of proteoforms due to post-translational modifications (PTMs) or protein-protein interactions (PPIs). MALDI-MS and MALDI tandem mass spectrometry (MS/MS) have been developed as analytical techniques to study small and large molecules, offering picomole to femtomole sensitivity and enabling the direct analysis of biological samples, such as biofluids, solid tissues, tissue/cell homogenates, and cell culture lysates, with a minimized procedure of sample preparation. In the last decades, structural identification of peptides and proteins achieved by MALDI-MS/MS helped researchers and clinicians to decipher molecular function, biological process, cellular component, and related pathways of the gene products as well as their involvement in pathogenesis of diseases. In this review, we highlight the applications of MALDI ionization source and tandem approaches for MS for analyzing biomedical relevant peptides and proteins. Furthermore, one of the most relevant applications of MALDI-MS/MS is to provide "molecular pictures", which offer in situ information about molecular weight proteins without labeling of potential targets. Histology-directed MALDI-mass spectrometry imaging (MSI) uses MALDI-ToF/ToF or other MALDI tandem mass spectrometers for accurate sequence analysis of peptide biomarkers and biological active compounds directly in tissues, to assure complementary and essential spatial data compared with those obtained by LC-ESI-MS/MS technique.

Structural Characterization of a New Collagen Biomimetic Octapeptide with Nanoscale Self-Assembly Potential: Experimental and Theoretical Approaches.

Bioinspired peptides are attractive biomolecules which can improve our understanding of self-assembly processes for rational design of new peptide-based materials. Herein, a new amidated peptide FRSAPFIE (FRS), based on a sequence present in human collagen, was synthesized, characterized by mass spectrometry and subjected to self-assembling investigations. The optimal conditions for self-assembly were disclosed by dynamic light scattering at 32 °C and a peptide concentration of 0.51 %. In addition, AFM studies revealed ellipsoidal FRS shapes with an area between 0.8 and 3.1 µm2 . The ability of self-assembly was also proved using FAD dye as extrinsic fluorescence reporter. According to the theoretical analysis, the FRS peptide tends to form a bundle-type association, with a type of fibrillary tangles particle. Altogether, our findings address new challenges regarding the FRS peptide which can be used in further self-assembly studies to design biocompatible drug-delivery platforms.

A Proteomic Approach to Identify Zein Proteins upon Eco-Friendly Ultrasound-Based Extraction.

Zein is a type of prolamin storage protein that has a variety of biomedical and industrial applications. Due to the considerable genetic variability and polyploidity of the starting material, as well as the extraction methods used, the characterization of the protein composition of zein requires a combination of different analytical processes. Therefore, we combined modern analytical methods such as mass spectrometry (MS), Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), atomic force microscopy (AFM), or Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) for a better characterization of the extracted zein. In this study, we present an enhanced eco-friendly extraction method, including grinding and sieving corn seeds, for prolamins proteins using an ultrasonic extraction methodology. The use of an ultrasonic homogenizer, 65% ethanol extraction buffer, and 710 µm maize granulation yielded the highest protein extraction from all experimental conditions we employed. An SDS PAGE analysis of the extracted zein protein mainly revealed two intense bands of approximatively 20 and 23 kDa, suggesting that the extracted zein was mostly α-zein monomer. Additionally, MS analysis revealed as a main component the α-zein PMS2 (Uniprot accession no. P24450) type protein in the maize flour extract. Moreover, AFM studies show that extracting zein with a 65% ethanol and a 710 µm granulation yields a homogeneous content that could allow these proteins to be employed in future medical applications. This research leads to a better understanding of zeins content critical for developing new applications of zein in food and pharmaceutical industries, such as biocompatible medical vehicles based on polyplexes complex nanoparticles of zein with antimicrobial or drug delivery properties.