Highlights • Single-pot workflow for manual or automated enrichment of N-terminal peptides. • Sensitive enrichment of protein N termini from 10,000 cells or 2 g crude proteome. • Data independent acquisition improves precision of peptide level quantification. • First degradomic analyses of sorted immune cells, single seedlings, and mitochondria from patient cells.

The nonlinear signal response of electrospray ionization (ESI) presents a critical limitation for mass spectrometry (MS)-based quantitative analysis. In the field of metabolomics research, this issue has largely remained unaddressed; MS signal intensities are usually directly used to calculate fold changes for quantitative comparison. In this work, we demonstrate that, due to the nonlinear ESI response, signal intensity ratios of a metabolic feature calculated between two samples may not reflect their real metabolic concentration ratios (i.e., fold-change compression), implying that conventional fold-change calculations directly using MS signal intensities can be misleading. In this regard, we developed a quality control (QC) sample-based signal calibration workflow to overcome the quantitative bias caused by the nonlinear ESI response. In this workflow, calibration curves for every metabolic feature are first established using a QC sample injected in serial injection volumes. The MS signals of each metabolic feature are then calibrated to their equivalent QC injection volumes for comparative analysis. We demonstrated this novel workflow in a targeted metabolite analysis, showing that the accuracy of fold-change calculations can be significantly improved. Furthermore, in a metabolomic comparison of the bone marrow interstitial fluid samples from leukemia patients before and after chemotherapy, an additional 59 significant metabolic features were found with fold changes larger than 1.5, and an additional 97 significant metabolic features had fold changes corrected by more than 0.1. This work enables high-quality quantitative analysis in untargeted metabolomics, thus providing more confident biological hypotheses generation.

The high affinity of biotin to streptavidin has made it one of the most widely used affinity tags in proteomics. Early methods used biotin for enrichment alone and mostly ignored the biotin labeled peptide. Recent advances in labeling led to an increase in biotinylation efficiency and shifted the interest to detection of the site of biotinylation. This increased confidence in identification and provides additional structural information yet it requires efficient release of the biotinylated protein/peptide and sensitive separation and detection of biotinylated peptides by LC-MS/MS. Despite its long use in affinity proteomics the effect of biotinylation on the chromatographic, ionization, and fragmentation behaviour and ultimate detection of peptides is not well understood. To address this we compare two commercially-available biotin labels EZ-Link Sulfo-NHS-Biotin and Sulfo-NHS-SS-Biotin, the latter one containing a labile linker to efficiently release biotin to determine the effects of peptide modification on peptide detection. We describe an increase of hydrophobicity and charge reduction with increasing number of biotin labels attached. Based on our data we recommend gradient optimization to account for more hydrophobic biotinylated peptides and include singly charged precursors to account for charge reduction by biotin.

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.

Murine xenografts of pediatric leukemia are known to accurately recapitulate genomic aberrations. How this translates to the functional capacity of the proteome is unknown. Here, we studied global protein abundance, phosphorylation, and proteolytic processing in 11 pediatric B-and T-cell acute lymphoblastic leukemia patients and 19 corresponding xenografts. Protein level differences that stratified pediatric disease subtypes at diagnostic and relapse stages were largely recapitulated in xenograft models. Patient xenografts lacked multiple human leukocyte antigens, and complement proteins, and presented incomplete response mechanisms to the host immune system which is absent in the murine model. The dominant expression of MKI67 and cell cycle proteins indicated a high proliferative capacity of xenografted cells residing in the spleen. Structural genomic changes and mutations found in patients were reflected at the protein level. The post-translational modification landscape is shaped by leukemia type and host and only to a limited degree by the patient of origin. This study portrays how genomic and host factors shape protein and post-translational modification landscapes differently, and confirms murine patient-derived xenograft as competent model system while highlighting important areas of diverging biology.

Protein N-termini reveal fundamental regulatory mechanisms and their perturbation in disease.Current terminome identification approaches are limited to whole organs or expandable cultured cells. We present a robust, sensitive, scalable and automatable method for system-wide identification of thousands of N-termini from minute samples. Identification of distinct Nterminal profiles in sorted immune cells, subcellular compartments, clinical biopsies, plasma from pediatric cancer patients, and protease substrates in Arabidopsis seedlings demonstrate broad applicability.

Background The bone marrow is the place of hematopoiesis with a microenvironment that supports lifelong maintenance of stem cells and high proliferation. It is not surprising that this environment is also favourable for malignant cells emerging in the bone marrow or metastasizing to it. While the cellular composition of the bone marrow microenvironment has been extensively studied, the extracellular matrix and interstitial fluid components have received little attention. Since the sinusoids connect the bone marrow interstitial fluid to the circulation, it is often considered to have the same composition as peripheral blood plasma. Stark differences in the cellular composition of the bone marrow and peripheral blood with different secretory capacities would however suggest profound differences. Methods In this study we set out to better define if and how the bone marrow interstitial fluid (BMIF) compares to the peripheral blood plasma (PBP) and how both are remodeled during chemotherapy. We applied a multi-omic strategy to quantify the metabolite, lipid and protein components as well as the proteolytic modification of proteins to gain a comprehensive understanding of the two compartments. Results We found that the bone marrow interstitial fluid is clearly distinct from peripheral blood plasma, both during active pediatric acute lymphoblastic leukemia and following induction chemotherapy. Either compartment was shaped differently by active leukemia, with the bone marrow interstitial fluid being rich in extracellular vesicle components and showing protease dysregulation while the peripheral blood plasma showed elevation of immune regulatory proteins. Following chemotherapy, the BMIF showed signs of cellular remodeling and impaired innate immune activation while the peripheral blood plasma was characterized by restored lipid homeostasis. Conclusion This study provides a comprehensive examination of the fluid portion of the acute lymphoblastic leukemia microenvironment and finds the contribution of either microenvironment to tumourigenesis. Supplementary Information The online version contains supplementary material available at 10.1186/s40164-022-00310-0.

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