PDX models reflect the proteome landscape of pediatric acute lymphoblastic leukemia but divert in select pathways.

BACKGROUND: Murine xenografts of pediatric leukemia accurately recapitulate genomic aberrations. How this translates to the functional capacity of cells remains unclear. Here, we studied global protein abundance, phosphorylation, and protein maturation by proteolytic processing in 11 pediatric B- and T- cell ALL patients and 19 corresponding xenografts. METHODS: Xenograft models were generated for each pediatric patient leukemia. Mass spectrometry-based methods were used to investigate global protein abundance, protein phosphorylation, and limited proteolysis in paired patient and xenografted pediatric acute B- and T- cell lymphocytic leukemia, as well as in pediatric leukemia cell lines. Targeted next-generation sequencing was utilized to examine genetic abnormalities in patients and in corresponding xenografts. Bioinformatic and statistical analysis were performed to identify functional mechanisms associated with proteins and protein post-translational modifications. RESULTS: Overall, we found xenograft proteomes to be most equivalent with their patient of origin. Protein level differences that stratified disease subtypes at diagnostic and relapse stages were largely recapitulated in xenografts. As expected, PDXs lacked multiple human leukocyte antigens and complement proteins. We found increased expression of cell cycle proteins indicating a high proliferative capacity of xenografted cells. Structural genomic changes and mutations were reflected at the protein level in patients. In contrast, the post-translational modification landscape was shaped by leukemia type and host and only to a limited degree by the patient of origin. Of 201 known pediatric oncogenic drivers and drug-targetable proteins, the KMT2 protein family showed consistently high variability between patient and corresponding xenografts. Comprehensive N terminomics revealed deregulated proteolytic processing in leukemic cells, in particular from caspase-driven cleavages found in patient cells. CONCLUSION: Genomic and host factors shape protein and post-translational modification landscapes differently. This study highlights select areas of diverging biology while confirming murine patient-derived xenografts as a generally accurate model system.

Sensitive Determination of Proteolytic Proteoforms in Limited Microscale Proteome Samples.

Protein N termini unambiguously identify truncated, alternatively translated or modified proteoforms with distinct functions and reveal perturbations in disease. Selective enrichment of N-terminal peptides is necessary to achieve proteome-wide coverage for unbiased identification of site-specific regulatory proteolytic processing and protease substrates. However, many proteolytic processes are strictly confined in time and space and therefore can only be analyzed in minute samples that provide insufficient starting material for current enrichment protocols. Here we present High-efficiency Undecanal-based N Termini EnRichment (HUNTER), a robust, sensitive and scalable method for the analysis of previously inaccessible microscale samples. HUNTER achieved identification of >1000 N termini from as little as 2 µg raw HeLa cell lysate. Broad applicability is demonstrated by the first N-terminome analysis of sorted human primary immune cells and enriched mitochondrial fractions from pediatric cancer patients, as well as protease substrate identification from individual Arabidopsis thaliana wild type and Vacuolar Processing Enzyme-deficient mutant seedlings. We further implemented the workflow on a liquid handling system and demonstrate the feasibility of clinical degradomics by automated processing of liquid biopsies from pediatric cancer patients.

Optical disc technology-enabled analytical devices: from hardware modification to digitized molecular detection.

Beyond their essential applications in portable data storage for the past 30 years, optical discs and corresponding recording/reading technologies have been extensively explored with the ultimate goal of creating novel analytical tools for on-site chemical analysis and point-of-care (POC) medical diagnosis. In particular, the disc media (CD, DVD, and BD) are proven to be inexpensive and versatile substrate materials for the preparation of various biochips and microfluidic systems; conventional computer drives and disc players are widely adapted for biochip signal reading and microscopic imaging. Herein we provide an overview of such optical disc technology-enabled analytical devices, e.g., integrated systems developed from specifically fabricated analog disks, modified optical drives, or adapted software algorithms.

Blu-ray Technology-Based Quantitative Assays for Cardiac Markers: From Disc Activation to Multiplex Detection.

Acute myocardial infarction (AMI) is the leading cause of mortality and morbidity globally. To reduce the number of mortalities, reliable and rapid point-of-care (POC) diagnosis of AMI is extremely critical. We herein present a Blu-ray technology-based assay platform for multiplex cardiac biomarker detection; not only off-the-shelf Blu-ray discs (BDs) were adapted as substrates to prepare standard immunoassays and DNA aptamer/antibody hybrid assays for the three key cardiac marker proteins (myoglobin, troponin I, and C-creative protein) but also an unmodified optical drive was directly employed to read the assay results digitally. In particular, we have shown that all three cardiac markers can be quantitated in their respective physiological ranges of interest, and the detection limits achieved are comparable with conventional enzyme-linked immunosorbent assay (ELISA) kits. The Blu-ray assay platform was further validated by measuring real-world samples and establishing a linear correlation with the simultaneously obtained ELISA data. Without the need to modify either the hardware (Blu-ray discs and optical drives) or the software driver, this assay-on-a-BD technique promises to be a low-cost user-friendly quantitative tool for on-site chemical analysis and POC medical diagnosis.

Magnetic Droplet Microfluidics as a Platform for the Concentration of [18F]Fluoride and Radiosynthesis of Sulfonyl [18F]Fluoride.

The radioisotope 18F is often considered the best choice for positron emission tomography (PET) owing to its desirable chemical and radiochemical properties. However, nucleophilic 18F-fluorination of large, water-soluble biomolecules, based on C-F bond formation, has traditionally been difficult. Thus, several aqueous fluorination approaches that offer significant versatility in radiopharmaceutical synthesis with sensitive targeting vectors have been developed. Furthermore, because 18F decays rapidly, production of these 18F-labeled compounds requires an automated process to reduce production time, reduce radiation exposure, and minimize losses due to the transfer of reagents during tracer synthesis. Herein, we report the use of magnetic droplet microfluidics (MDM) as a means to concentrate [18F]fluoride from the cyclotron target solution, followed by the synthesis of an 18F-labeled compound on a microfluidic platform. Using this method, we have demonstrated 18F preconcentration in a small-volume droplet through the use of anion exchanging magnetic particles. By using MDM, the preconcentration step took approximately 5 min, and the [18F]fluoride solution was preconcentrated by 15-fold. After the preconcentration step, an 18F-labeling reaction was performed on the MDM platform using the S-F bond formation in aqueous conditions to produce an arylsulfonyl [18F]fluoride compound which can be used as a prosthetic group to label PET targeting ligands. The high radiochemical purity of 95±1% was comparable to the 96% previously reported using a conventional method. In addition, when MDM was used, the total synthesis time was improved to 15 min with lower reagent volumes (50-60 µL) used.

DVD technology-based molecular diagnosis platform: quantitative pregnancy test on a disc.

A diagnosis platform based entirely on DVD technology was developed for on-site quantitation of molecular analytes of interest, e.g., human chorionic gonadotropin (hCG) in urine samples ("quantitative pregnancy test on a disc"). An hCG-specific monoclonal antibody-binding assay prepared on a regular DVD-R was labeled with nanogold-streptavidin conjugates for signal enhancement with a customized silver-staining protocol. An unmodified, conventional computer optical drive was used for assay reading, and free disc-quality analysis software for data processing. The performance (sensitivity and selectivity) of this DVD assay is comparable to that of well-established colorimetric methods (determination of optical darkness ratios) and standard enzyme-linked immunosorbent assays (ELISA). As validated by examining its linear correlation with the ELISA results on the same set of samples, the DVD assay promises to be a low-cost, multiplex, point-of-care (POC) diagnostic tool for physicians and even for individuals at home, producing prompt results.