Hexagonal Mesoporous Silica as a Rapid, Efficient and Versatile Tool for MALDI-TOF MS Sample Preparation in Clinical Peptidomics Analysis: A Pilot Study

Abstract

Improvement in high-throughput MALDI-TOF MS analysis requires practical and efficient sample preparation protocols for high acquisition rates. The use of hexagonal mesoporous silica (HMS) sorbents in combination with MALDI-TOF MS was explored as a versatile tool for peptidomic profiling of clinical specimens difficult to process, but considered important sources of disease biomarkers: synovial fluid and sputum. A rapid and efficient procedure, based on dispersive solid-phase extraction of peptides using commercially available wormhole mesostructured HMS, was tested for: a) pre-concentration of standard peptides in serially diluted solution up to the sub-nanomolar range; b) peptidome profiling of sputum and synovial fluid. The use of HMS, as dispersed sponges, significantly amplified the peptidic repertoire of sputum and synovial fluid by excluding from the adsorptive process large size proteins, which mask and/or suppress peptidome signals. The protocol proposed, as dispersive solid phase extraction, ensures good analytical performances. Moreover, it is economical and rapid, as it avoids the use of less reproducible and prolonged sample preparation procedures, such as the use of ultrafiltration filter devices. These findings may contribute to defining a high-throughput screening MS-based platform for monitoring key peptidic features of difficult to analyse bodily fluids in a clinical setting.

Proteomic Analysis of Human Sputum for the Diagnosis of Lung Disorders: Where Are We Today?

“…This concept was reaffirmed by O'Neil et al [ 34 ] who, in a review article focused on the importance of identifying subgroups of disease entities to establish appropriate biomarkers and to enhance the understanding of underlying mechanisms in each subgroup, argued that advanced proteomics techniques have the inherent ability to provide a significant contribution to this cause. The work by Terracciano et al [ 9 , 11 , 12 , 35 ] seems to fit this perspective. In fact, to elucidate the mechanisms underlying the pathobiology of COPD and asthma, they investigated the proteomic/peptidomic profiles of sputum from patients affected by these two disorders.…”

“…In detail, six signals corresponding to three human α-defensins and three C-terminal amidated peptides (one of which was phosphorylated on serine), emerged as potential diagnostic peptidyl patterns able to differentiate these inflammatory airway diseases. Remaining within the scope of peptidomics, with the aim of improving the effectiveness of sample preparation protocols, these authors explored the benefits provided by another mesoporous silica sorbent [ 35 ]. Characterized by higher stability and thicker pore walls than the previous one [ 9 ], this material proved to be a versatile tool for peptidomic profiling of both sputum and synovial fluid, chosen as clinical sources of potential lung disease biomarkers.…”

MALDI MS-Based Investigations for SARS-CoV-2 Detection

“…MALDI-TOF MS allows for the analysis of hundreds of proteins or other biomolecules in a single spectrum and has been used in several diagnostic applications in recent years, including: (i) the study of the distributions of particular biochemical compounds on tissues (MALDI-based imaging MS) [42,43]; (ii) the analysis of nucleic acids for genotyping single nucleotide polymorphisms (SNPs) [44]; (iii) the profiling of biological samples to aid in the discovery of new of disease biomarkers in exploratory studies with diagnostic applicability. Such biomarkers might be used as indicators of progress or the severity of diseases/disorders [45][46][47][48][49][50][51], and for (iv) the identification, characterization, and typing of bacteria, yeasts, and viruses [52][53][54][55][56].…”

“…diseases/disorders [45][46][47][48][49][50][51], and for (iv) the identification, characterization, and typing of bacteria, yeasts, and viruses [52][53][54][55][56]. The ions are accelerated toward a detector, which amplifies ion signals and converts them into a mass spectrum.…”

Gingival Crevicular Fluid Peptidome Profiling in Healthy and in Periodontal Diseases

“…It is interesting to underline that the use of acidic buffer in peptidomics investigations might turn useful also for other reasons. In fact, high molecular weight proteins such as mucins, lactoferrin, α-amylases, and myeloperoxidases, found to be present in GCF [ 14 , 30 , 31 , 33 , 34 , 36 , 37 , 38 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ] and which could hinder the detection of peptidic components [ 105 ], are insoluble in acidic conditions [ 27 ]. Studies on peptidome stability of GCF samples with PIC supplementation at neutral or basic pH are not yet reported in literature.…”