Isolation and Proteomic Analysis of Mouse Serum Small Extracellular Vesicles for Individual Subject Analysis.

Proteomics characterization of blood and circulating material has been extensively explored for the study of pathological states. In particular, circulating small extracellular vesicles (sEV, diameter: 30-150 nm) are known to play an important role in intercellular communication processes, and proteomics profiling has been explored to develop minimally invasive assays for disease monitoring and diagnosis. Due to the genetic and physiological similarities between the two species, and also on account of their shorter life span and rapid disease progression, rodent models are the most commonly used animal model for many human diseases. Such models have provided invaluable insight into the molecular mechanisms of disease progression, candidate drug efficacy, therapy monitoring, and biomarkers research.Longitudinal investigations, in which individuals are monitored over periods of time, are more able to resolve molecular changes during disease progression because they circumvent the inter-individual variation. Longitudinal investigations of rodent models are challenging because of the limited amount of blood that can be withdrawn at each time; the American Association of Veterinary Science stipulates that fortnightly sampling should be limited to a maximum of 10% of the total blood volume. For adult mice this corresponds to approximately 75 µL of serum. We developed an approach for the isolation and characterization of serum sEV proteins from just 50 µL of serum, for longitudinal studies of disease mouse models. This chapter describes in detail the steps and considerations involved in the sEV isolation, morphological characterization, and proteome profiling by mass spectrometry.

ANKRD44 Gene Silencing: A Putative Role in Trastuzumab Resistance in Her2-Like Breast Cancer.

Trastuzumab is an effective therapeutic treatment for Her2-like breast cancer; despite this most of these tumors develop resistance to therapy due to specific gene mutations or alterations in gene expression. Understanding the mechanisms of resistance to Trastuzumab could be a useful tool in order to identify combinations of drugs that elude resistance and allow a better response for the treated patients. Twelve primary biopsies of Her2+/hormone receptor negative (ER-/PgR-) breast cancer patients were selected based on the specific response to neoadjuvant therapy with Trastuzumab and their whole exome was sequenced leading to the identification of 18 informative gene mutations that discriminate patients selectively based on response to treatment. Among these genes, we focused on the study of the ANKRD44 gene to understand its role in the mechanism of resistance to Trastuzumab. The ANKRD44 gene was silenced in Her2-like breast cancer cell line (BT474), obtaining a partially Trastuzumab-resistant breast cancer cell line that constitutively activates the NF-kb protein via the TAK1/AKT pathway. Following this activation an increase in the level of glycolysis in resistant cells is promoted, also confirmed by the up-regulation of the LDHB protein and by an increased TROP2 protein expression, found generally associated with aggressive tumors. These results allow us to consider the ANKRD44 gene as a potential gene involved in Trastuzumab resistance.

Quantitative Microproteomics Based Characterization of the Central and Peripheral Nervous System of a Mouse Model of Krabbe Disease.

Krabbe disease is a rare, childhood lysosomal storage disorder caused by a deficiency of galactosylceramide beta-galactosidase (GALC). The major effect of GALC deficiency is the accumulation of psychosine in the nervous system and widespread degeneration of oligodendrocytes and Schwann cells, causing rapid demyelination. The molecular mechanisms of Krabbe disease are not yet fully elucidated and a definite cure is still missing. Here we report the first in-depth characterization of the proteome of the Twitcher mouse, a spontaneous mouse model of Krabbe disease, to investigate the proteome changes in the Central and Peripheral Nervous System. We applied a TMT-based workflow to compare the proteomes of the corpus callosum, motor cortex and sciatic nerves of littermate homozygous Twitcher and wild-type mice. More than 400 protein groups exhibited differences in expression and included proteins involved in pathways that can be linked to Krabbe disease, such as inflammatory and defense response, lysosomal proteins accumulation, demyelination, reduced nervous system development and cell adhesion. These findings provide new insights on the molecular mechanisms of Krabbe disease, representing a starting point for future functional experiments to study the molecular pathogenesis of Krabbe disease. Data are available via ProteomeXchange with identifier PXD010594.

Mass Spectrometry Imaging, Laser Capture Microdissection, and LC-MS/MS of the Same Tissue Section.

Mass spectrometry imaging (MSI) is able to simultaneously record the distributions of hundreds of molecules directly from tissue. Rapid direct tissue analysis is essential for MSI in order to maintain spatial localization and acceptable measurement times. The absence of an explicit analyte separation/purification step means MSI lacks the depth of coverage of LC-MS/MS. In this work, we demonstrate how atmospheric pressure MALDI-MSI enables the same tissue section to be first analyzed by MSI, to identify regions of interest that exhibit distinct molecular signatures, followed by localized proteomics analysis using laser capture microdissection isolation and LC-MS/MS.

In-Source Decay and Pseudo-MS3 of Peptide and Protein Ions Using Liquid AP-MALDI.

Atmospheric pressure MALDI on a Q-Exactive instrument was optimized for in-source decay and pseudo-MS3. The dependence of AP-MALDI ISD on the MALDI liquid matrix was investigated for peptides and proteins. The liquid matrices enabled long-life ISD signal, and exhibited high fragment ion yield and signal stability. Extensive a-, b-, c-, y-, and z-type fragment series were observed depending on the matrix used but were most extensive with 2,5-DHB. Complete sequence coverage of small peptide and intact protein-terminus sequence tags were obtained and confirmed using HCD as a pseudo-MS3 method. Graphical Abstract ᅟ.

Set of Novel Automated Quantitative Microproteomics Protocols for Small Sample Amounts and Its Application to Kidney Tissue Substructures.

Here we assessed the ability of an automated sample preparation device equipped with disposable microcolumns to prepare mass-limited samples for high-sensitivity quantitative proteomics, using both label-free and isobaric labeling approaches. First, we compared peptide label-free quantification reproducibility for 1.5-150 µg of cell lysates and found that labware preconditioning was essential for reproducible quantification of <7.5 µg digest. Second, in-solution and on-column tandem mass tag (TMT) labeling protocols were compared and optimized for 1 µg of sample. Surprisingly, standard methods for in-solution and on-column labeling showed poor TMT labeling (50-85%); however, novel optimized and automated protocols restored efficient labeling to >98%. Third, compared with a single long gradient experiment, a simple robotized high-pH fractionation protocol using only 6 µg of starting material doubled the number of unique peptides and increased proteome coverage 1.43-fold. To facilitate the analysis of heterogeneous tissue samples, such as those obtained from laser capture microdissection, a modified BCA protein assay was developed that consumes and detects down to 15 ng of protein. As a proof-of-principle, the modular automated workflow was applied to 0.5 and 1 mm2 mouse kidney cortex and medulla microdissections to show the method's potential for real-life small sample sources and to create kidney substructure-specific proteomes.

Development and validation of a novel derivatization method for the determination of lactate in urine and saliva by liquid chromatography with UV and fluorescence detection.

We developed a novel and straightforward derivatization method for the determination of lactate by reversed phase high-performance liquid chromatography (RP-HPLC) with fluorescence and UV detection in biological matrices as urine and saliva. The derivatization of lactate was achieved employing 9-chloromethyl anthracene (9-CMA) as fluorescence reagent, which has never been previously used to obtain a lactate derivative. Lactate reacts with 9-CMA with high selectivity in a very short time, without requiring extraction procedures from the aqueous solution, and the reaction reaches 70% completion in 30 min. The ester derivative obtained can be easily determined by RP-HPLC with fluorescence detection at 410 nm (λ ex=365 nm) and UV detection at 365 nm. The method was also optimized in order to allow for the simultaneous determination of lactate and creatinine for the application to urine samples. The lactate calibration curve was linear in the investigated range 2 × 10(-4)-3 × 10(-2)mM and the limit of detection, calculated as three times the standard deviation of the blank divided by the calibration curve slope, was 50 nM for both fluorescence and UV detection. Intra-day and inter-day repeatability were lower than 5% and 6%, respectively. The method proposed was successfully applied to the analysis of urine and saliva samples.

Adenocarcinoma arising in an elderly patient's large ileal duplication.

Bowel duplications are rare congenital anomalies commonly found in pediatric patients; few cases may remain undetected until adulthood. Malignant carcinomatous changes are rare complications in intestinal duplications. An 88-year-old female patient was referred to our surgical unit with the diagnosis of a large abdominal mass. An explorative laparotomy was performed, revealing a large (22 × 11 cm) neoplasm strictly connected to the lowest ileal segment and completely filling the pelvis. Definitive histology revealed a moderately differentiated adenocarcinoma developing in a duplication of the terminal ileum. The hypothesis of a gastrointestinal duplication should be evaluated in the differential diagnosis of large, complex, indeterminate masses located in or near the bowel; the possibility of neoplasm within the duplication should be considered.

Jejuno-jejunal invagination due to intestinal melanoma.

Cutaneous melanoma is one of the most studied neoplastic lesions in biology and clinical oncology. It has been well documented that this type of neoplasm presents a high metastatic rate, and is able to involve nearly every tissue. Non-cutaneous melanoma represents an unusual pattern of melanoma, and the small intestine is an uncommon anatomic localization. Herein we report an extremely rare clinical case of a young woman affected by a bleeding jejunal melanoma, whose early clinical presentation was an intestinal invagination.