Reproducible Profiling Research Articles

Drug Permeability Profiling Using the Novel Permeapad® 96-Well Plate.

Here, first experiences with a prototype tool for high throughput (passive) permeability profiling, a 96-well plate comprising the Permeapad® membrane, are reported. The permeabilities of a set of drugs were determined and compared to published measures of oral absorption, such as human fraction absorbed (Fa) and in vitro permeability values obtained using other tools. The tool consists of a 96-well bottom and screen plate with the artificial, phospholipid-based barrier (Permeapad®) mounted between the plates' lower and upper compartments. The permeability of 14 model compounds including high- and low-absorption drugs, cationic, anionic, zwitterionic and neutral molecules, was determined by quantifying the compounds' transport over time, deriving the steady-state flux from the linear part of the cumulative curves and calculating the apparent permeability (Papp). The membrane structure was investigated in a high-resolution digital light microscope. The Permeapad® 96-well plate was found suited to distinguish high and low absorption drugs and yielded a hyperbolic correlation to Fa. The Papp values obtained were congruent with those determined with in-house prepared Permeapad® in the Franz cell set-up. Furthermore, good to excellent correlations were seen with Caco-2 permeability (R2= 0.70) and PAMPA permeability (R2= 0.89). Microscopic investigation of the Permeapad® barrier revealed the formation of phospholipid vesicles and myelin figures in aqueous environment. The Permeapad® 96-well plate permeation set-up is a promising new tool for rapid and reproducible passive permeability profiling.

Advancing serum peptidomic profiling by data-independent acquisition for clear-cell renal cell carcinoma detection and biomarker discovery

The analysis of serum endogenous peptides holds promise for disease study and drug discovery, whereas it is relatively unexplored given its challenges in analysis reproducibility and reliability. Here, we developed a streamlined detection platform for high-sensitive and reproducible serum peptidome profiling by data-independent acquisition (DIA) strategy. Compared to the classic data-dependent acquisition (DDA), our developed DIA approach can quantify almost twice the number of peptides with half the median coefficients of variation detected for DDA. The platform enables reproducible profiling of thousands of peptides and their post-translational modifications after simple sample preparation. The developed platform was subsequently utilized in the serum peptidome study of clear-cell renal cell carcinoma (ccRCC). A total of 31 ccRCC patients and 31 healthy volunteers were enrolled. Significant differences in serum peptidome patterns were observed between the two groups, allowing us to distinguish ccRCC patients from healthy volunteers clearly. A total of 833 modified peptides were found significantly changed in the ccRCC patients. The study demonstrated the high potential of serum peptidome in cancer detection and the feasibility and advantage of applying the DIA-based platform on large-scale serum peptidomic analysis for biomarker discovery. SignificanceSerum peptidomic study proves to be challenging given its low abundance and instability of endogenous peptides. In this study, we developed a fast, reproducible and accurate detection platform by DIA-based MS method for streamlined serum peptidome profiling. The developed platform was then utilized in the serum peptidome study of ccRCC. To our knowledge, this is the first report to apply DIA strategy to disease related peptidomic studies. The large difference in serum peptidome profiles enabled us to distinguish ccRCC patients from healthy volunteers clearly, illuminating the great potential of serum peptidome in cancer diagnosis. The discovered significantly changed peptides provided a better understanding of the pathophysiological changes in ccRCC.

A mouse SWATH-mass spectrometry reference spectral library enables deconvolution of species-specific proteomic alterations in human tumour xenografts.

ABSTRACTSWATH-mass spectrometry (MS) enables accurate and reproducible proteomic profiling in multiple model organisms including the mouse. Here, we present a comprehensive mouse reference spectral library (MouseRefSWATH) that permits quantification of up to 10,597 proteins (62.2% of the mouse proteome) by SWATH-MS. We exploit MouseRefSWATH to develop an analytical pipeline for species-specific deconvolution of proteomic alterations in human tumour xenografts (XenoSWATH). This method overcomes the challenge of high sequence similarity between mouse and human proteins, facilitating the study of host microenvironment-tumour interactions from ‘bulk tumour’ measurements. We apply the XenoSWATH pipeline to characterize an intraductal xenograft model of breast ductal carcinoma in situ and uncover complex regulation consistent with stromal reprogramming, where the modulation of cell migration pathways is not restricted to tumour cells but also operates in the mouse stroma upon progression to invasive disease. MouseRefSWATH and XenoSWATH open new opportunities for in-depth and reproducible proteomic assessment to address wide-ranging biological questions involving this important model organism.

Machine Learning with Optical Phase Signatures for Phenotypic Profiling of Cell Lines.

Robust and reproducible profiling of cell lines is essential for phenotypic screening assays. The goals of this study were to determine robust and reproducible optical phase signatures of cell lines for classification with machine learning and to correlate optical phase parameters to motile behavior. Digital holographic microscopy (DHM) reconstructed phase maps of cells from two pairs of cancer and non-cancer cell lines. Seventeen image parameters were extracted from each cell's phase map, used for linear support vector machine learning, and correlated to scratch wound closure and Boyden chamber chemotaxis. The classification accuracy was between 90% and 100% for the six pairwise cell line comparisons. Several phase parameters correlated with wound closure rate and chemotaxis across the four cell lines. The level of cell confluence in culture affected phase parameters in all cell lines tested. Results indicate that optical phase features of cell lines are a robust set of quantitative data of potential utility for phenotypic screening and prediction of motile behavior. © 2019 International Society for Advancement of Cytometry.

Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution

The breadth and importance of RNA modifications are growing rapidly as modified ribonucleotides can impact the sequence, structure, function, stability, and fate of RNAs and their interactions with other molecules. Therefore, knowing cellular RNA modifications at single-base resolution could provide important information regarding cell status and fate. A current major limitation is the lack of methods that allow the reproducible profiling of multiple modifications simultaneously, transcriptome-wide and at single-base resolution. Here we developed RBS-Seq, a modification of RNA bisulfite sequencing that enables the sensitive and simultaneous detection of m5C, Ψ, and m1A at single-base resolution transcriptome-wide. With RBS-Seq, m5C and m1A are accurately detected based on known signature base mismatches and are detected here simultaneously along with Ψ sites that show a 1-2 base deletion. Structural analyses revealed the mechanism underlying the deletion signature, which involves Ψ-monobisulfite adduction, heat-induced ribose ring opening, and Mg2+-assisted reorientation, causing base-skipping during cDNA synthesis. Detection of each of these modifications through a unique chemistry allows high-precision mapping of all three modifications within the same RNA molecule, enabling covariation studies. Application of RBS-Seq on HeLa RNA revealed almost all known m5C, m1A, and ψ sites in tRNAs and rRNAs and provided hundreds of new m5C and Ψ sites in noncoding RNAs and mRNAs. However, our results diverge greatly from earlier work, suggesting ∼10-fold fewer m5C sites in noncoding and coding RNAs and the absence of substantial m1A in mRNAs. Taken together, the approaches and refined datasets in this work will greatly enable future epitranscriptome studies.

Comprehensive and quantitative analysis of white and brown adipose tissue by shotgun lipidomics

ObjectiveShotgun lipidomics enables an extensive analysis of lipids from tissues and fluids. Each specimen requires appropriate extraction and processing procedures to ensure good coverage and reproducible quantification of the lipidome. Adipose tissue (AT) has become a research focus with regard to its involvement in obesity-related pathologies. However, the quantification of the AT lipidome is particularly challenging due to the predominance of triacylglycerides, which elicit high ion suppression of the remaining lipid classes. MethodsWe present a new and validated method for shotgun lipidomics of AT, which tailors the lipid extraction procedure to the target specimen and features high reproducibility with a linear dynamic range of at least 4 orders of magnitude for all lipid classes. ResultsUtilizing this method, we observed tissue-specific and diet-related differences in three AT types (brown, gonadal, inguinal subcutaneous) from lean and obese mice. Brown AT exhibited a distinct lipidomic profile with the greatest lipid class diversity and responded to high-fat diet by altering its lipid composition, which shifted towards that of white AT. Moreover, diet-induced obesity promoted an overall remodeling of the lipidome, where all three AT types featured a significant increase in longer and more unsaturated triacylglyceride and phospholipid species. ConclusionsThe here presented method facilitates reproducible systematic lipidomic profiling of AT and could be integrated with further –omics approaches used in (pre-) clinical research, in order to advance the understanding of the molecular metabolic dynamics involved in the pathogenesis of obesity-associated disorders.

Systematic miRNome profiling reveals differential microRNAs in transgenic maize metabolism

BackgroundWhile some genetically modified organisms (GMOs) are created to produce new double-stranded RNA molecules (dsRNA), in others, such molecules may occur as an unintended effect of the genetic engineering process. Furthermore, GMOs might produce naturally occurring dsRNA molecules in higher or lower quantities than its non-transgenic counterpart. This study is the first to use high-throughput technology to characterize the miRNome of commercialized GM maize events and to investigate potential alterations in miRNA regulatory networks.ResultsThirteen different conserved miRNAs were found to be dys-regulated in GM samples. The insecticide Bt GM variety had the most distinct miRNome. These miRNAs target a range of endogenous transcripts, such as transcription factors and nucleic acid binding domains, which play key molecular functions in basic genetic regulation. In addition, we have identified 20 potential novel miRNAs with target transcripts involved in lipid metabolism in maize. isomiRs were also found in 96 conserved miRNAs sequences, as well as potential transgenic miRNA sequences, which both can be a source of potential off-target effects in the plant genome. We have also provided information on technical limitations and when to carry on additional in vivo experimental testing.ConclusionsThese findings do not reveal hazards per se but show that robust and reproducible miRNA profiling technique can strengthen the assessment of risk by detecting any new intended and unintended dsRNA molecules, regardless of the outcome, at any stage of GMO development.

BAMP-28 Antimicrobial Peptide Against Different MALDI Biotype of Carbapenam Resistant Enterobacteriaceae

Worldwide emergence of Carbapenam resistance in Enterobacteriaceae (CRE) are increasing globally and becoming a severe public health issue. Infections caused by CRE have limited treatment options and have been associated with high mortality rates. Due to their unique mode of action, antimicrobial peptides are novel alternatives to traditional antibiotics for tackling the issue of bacterial multidrug resistance. An easy, rapid and accurate detection of 72 clinically CRE isolates using a MALDI–TOF MS was additionally developed. The CRE isolates belonging to 33 Carbapenam-resistant Klebsiella pneumoniae, 17 Carbapenam-resistant Escherichia coli, 16 Carbapenam-resistant Enterobacter cloacae and 6 Carbapenam-resistant Citrobacter freundii carrying blaNDM-1 were definitely discriminated from reference genotype strain by MALDI–TOF MS. This rapid, accurate, and reproducible peptide signature profiling technology could have new implications in laboratory-based high-throughput differentiation of extensive libraries of Carbapenam resistant Enterobacteriaceae. Antibacterial activity of 9 short novel peptides against these CRE isolates were investigated. Although neither synthetic peptides induced significant hemolysis, or showed cytotoxic on Vero cell, only BAMP-28 peptide inhibited growth of K. pneumoniae, E. coli, C. freundii and E. cloacae with MIC50 of 18–40, 20–40, 16–25 and 18–36 µM, respectively. In conclusion, MALDI–TOF MS can be used to screen for Carbapenam resistance in K. pneumoniae, E. coli, E. cloacae and C. freundii. Interestingly, BMAP-28 peptide had acceptable effect on Carbapenam resistant Enterobacteriaceae including K. pneumoniae, E. coli, C. freundii and E. Cloacae isolates with less toxicity.

Proteome-based systems biology in chronic pain

Chronic pain represents a major medical challenge in the 21st century. Enormous efforts have been invested towards deciphering the complexity of chronic pain from different angles (molecular, physiological, psychosocial, and behavioral) in both preclinical and clinical settings. While progress has been made, our understanding of the underlying mechanisms of chronic pain remains insufficient. Consequently, chronic pain treatment is often inadequate. It lacks efficacy in most patients and is associated with detrimental side effects – a situation which calls for urgent changes in pain research and management.In this review we propose that protein-centric systems biology can significantly contribute to pain research. This approach may introduce the long-awaited paradigm shift in pain research from single targets to multidimensional cellular networks. We critically discuss how recent advances in reproducible and comprehensive proteome profiling can be exploited by pain researchers in the following ways: to gain mechanistic insights into chronic pain and its diverse forms, to facilitate clinical trials and the search for new drug targets, and to objectively assess chronic pain and its stages in individual patients by defining so-called protein disease signatures (PDS).We feel that the integration of proteomics into the toolbox of pain researchers and physicians alike will open new avenues towards a better understanding and management of chronic pain. Significance statementThe immense challenges associated with chronic pain call for urgent changes in pain research and management. Here, we highlight the enormous potential of a proteome-based systems biology approach for advancing our understanding of chronic pain from a mechanistic, translational and clinical angle.

GC-MS profiling of leukemia cells: an optimized preparation protocol for the intracellular metabolome

A preparation protocol for the robust and reproducible metabolome profiling of leukemia cells with relatively high coverage has been developed.

Vio®Dyes meet REAfinity - High Resolution Antibody-Fluorochrome Conjugates for Multi-Parameter Flow Cytometry Analysis

Abstract State-of-the-art sensitivity and target specificity of fluorochrome-conjugated antibodies limit applications in flow cytometry. Identification of cell subsets can be challenging due to low expression levels of surface and intracellular markers. The combination of Vio®Dyes and REAfinity™ antibodies provides researchers with the most powerful and reliable tools for flow cytometric cells analysis. REAfinity™ are recombinant antibodies of human IgG1 isotype with a mutated region within the Fc part, which prevents Fcγ receptor interactions. They are characterized by high antigen specificity and consistent quality compared to conventional hybridoma-derived monoclonal antibodies. Vio®Dyes, e.g. Vio®515, VioBright®515, Vio®667, VioBright®667, and PE-Vio®615, allow due to their excellent fluorescent properties the detection of low expressed markers and provide new choices for multi-parameter analysis. To demonstrate the potential of the combination of REAfinity™ antibodies and Vio®Dyes, we developed detection panels and protocols for the characterization of FOXP3+, HELIOS+, and CD45RA+ in CD4+ CD25+ CD127low/− human regulatory T cells (Tregs). Tregs were detected and enumerated in whole blood, PBMC, and in samples of magnetically enriched Tregs utilizing the newly developed reagents. In addition, we set-up new and fast staining protocols using 96-well plates or magnetic columns, which allow improved identification of Treg subsets. In summary, the portfolio of Vio®Dyes conjugated REAfinity and special protocols open up new ways for designing and implementing multicolor panels towards highly sensitive, reproducible, and rapid cell profiling approaches.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Utilization of Whole-Cell MALDI-TOF Mass Spectrometry to Differentiate Burkholderia pseudomallei Wild-Type and Constructed Mutants.

Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has been widely adopted as a useful technology in the identification and typing of microorganisms. This study employed the whole-cell MALDI-TOF MS to identify and differentiate wild-type and mutants containing constructed single gene mutations of Burkholderia pseudomallei, a pathogenic bacterium causing melioidosis disease in both humans and animals. Candidate biomarkers for the B. pseudomallei mutants, including rpoS, ppk, and bpsI isolates, were determined. Taxon-specific and clinical isolate-specific biomarkers of B. pseudomallei were consistently found and conserved across all average mass spectra. Cluster analysis of MALDI spectra of all isolates exhibited separate distribution. A total of twelve potential mass peaks discriminating between wild-type and mutant isolates were identified using ClinProTools analysis. Two peaks (m/z 2721 and 2748 Da) were specific for the rpoS isolate, three (m/z 3150, 3378, and 7994 Da) for ppk, and seven (m/z 3420, 3520, 3587, 3688, 4623, 4708, and 5450 Da) for bpsI. Our findings demonstrated that the rapid, accurate, and reproducible mass profiling technology could have new implications in laboratory-based rapid differentiation of extensive libraries of genetically altered bacteria.

Short-column anion-exchange chromatography for soil and peat humic substances profiling by step-wise gradient of high pH aqueous sodium ethylenediaminetetraacetate

Novel anion-exchange liquid chromatographic method with step gradient of aqueous EDTA4− based mobile phase elution has been developed to profile available Slovak soil humic substances and alkaline extracts of various soils. The method utilize short glass column (30mm×3mm) filled in with hydrolytically stable particles (60μm diameter) Separon HEMA-BIO 1000 having (diethylamino)ethyl functional groups. Step gradient was programmed by mixing mobile phase composed of aqueous solution of sodium EDTA (pH 12.0; 5mmolL−1) and mobile phase constituted of aqueous solution of sodium EDTA (pH 12.0, 500mmolL−1). The FLD of HSs was set to excitation wavelength 480nm and emission wavelength 530nm (λem). Separation mechanism was studied by use of selected aromatic acids related to humic acids with the aid of UV spectrophotometric detection at 280nm. The proposed method benefits from high ionic strength (I=5molL−1) of the end mobile phase buffer and provides high recovery of humic acids (98%). Accurate and reproducible profiling of studied humic substances, alkaline extracts of various types of soils enables straightforward characterization and differentiation of HSs in arable and forest soils. Selected model aromatic acids were used for separation mechanism elucidation.

Proteomic profiling of the retinas in a neonatal rat model of oxygen-induced retinopathy with a reproducible ion-current-based MS1 approach.

Investigation of the retina proteome during hypoxia-induced retinal neovascularization is valuable for understanding pathogenesis of retinopathy of prematurity (ROP). Here we employed a reproducible ion-current-based MS1 quantification approach (ICB) to explore the retinal proteomic changes in early stage of ROP in a rat model of oxygen-induced retinopathy (OIR). Retina proteins, which are rich in membrane proteins, were efficiently extracted by a detergent-cocktail and subjected to precipitation/on-pellet-digestion, followed by nano-LC-MS analysis on a 75-cm column with a 7-h gradient. The high reproducibility of sample preparation and chromatography separation enabled excellent peak alignment and contributed to the superior performance of ICB over parallel label-free approaches. In this study, sum-of-intensity with rejection was incorporated to determine the protein ratios. In total, 1325 unique protein groups were quantified from rat retinas (n = 4/group) with at least two distinct peptides at a protein FDR of 1%. Thirty-two significantly altered proteins were observed with confidence, and the elevated glial fibrillary acidic protein and decreased crystalline proteins in OIR retinas agree well with previous studies. Selected key alterations were further validated by Western blot analysis. Interestingly, Rab21/RhoA/ROCK2/moesin signaling pathway was found to be involved in retinal neovascularization of OIR. Moreover, highly elevated annexin A3, a potential angiogenic mediator, was observed in OIR retinas and may serve as a potential therapeutic target. In conclusion, reproducible ICB profiling enabled reliable discovery of many altered mediators and pathways in OIR retinas, thereby providing new insights into molecular mechanisms involved in pathogenesis of ROP.

Fluorescently activated cell sorting followed by microarray profiling of helper T cell subtypes from human peripheral blood.

BackgroundPeripheral blood samples have been subjected to comprehensive gene expression profiling to identify biomarkers for a wide range of diseases. However, blood samples include red blood cells, white blood cells, and platelets. White blood cells comprise polymorphonuclear leukocytes, monocytes, and various types of lymphocytes. Blood is not distinguishable, irrespective of whether the expression profiles reflect alterations in (a) gene expression patterns in each cell type or (b) the proportion of cell types in blood. CD4+ Th cells are classified into two functionally distinct subclasses, namely Th1 and Th2 cells, on the basis of the unique characteristics of their secreted cytokines and their roles in the immune system. Th1 and Th2 cells play an important role not only in the pathogenesis of human inflammatory, allergic, and autoimmune diseases, but also in diseases that are not considered to be immune or inflammatory disorders. However, analyses of minor cellular components such as CD4+ cell subpopulations have not been performed, partly because of the limited number of these cells in collected samples.Methodology/Principal FindingsWe describe fluorescently activated cell sorting followed by microarray (FACS–array) technology as a useful experimental strategy for characterizing the expression profiles of specific immune cells in the circulation. We performed reproducible gene expression profiling of Th1 and Th2, respectively. Our data suggest that this procedure provides reliable information on the gene expression profiles of certain small immune cell populations. Moreover, our data suggest that GZMK, GZMH, EOMES, IGFBP3, and STOM may be novel markers for distinguishing Th1 cells from Th2 cells, whereas IL17RB and CNTNAP1 can be Th2-specific markers.Conclusions/SignificanceOur approach may help in identifying aberrations and novel therapeutic or diagnostic targets for diseases that affect Th1 or Th2 responses and elucidating the involvement of a subpopulation of immune cells in some diseases.

Mass spectrometry in cancer biomarker research: a case for immunodepletion of abundant blood-derived proteins from clinical tissue specimens.

The discovery of clinically relevant cancer biomarkers using mass spectrometry (MS)-based proteomics has proven difficult, primarily because of the enormous dynamic range of blood-derived protein concentrations and the fact that the 22 most abundant blood-derived proteins constitute approximately 99% of the total plasma protein mass. Immunodepletion of clinical body fluid specimens (e.g., serum/plasma) for the removal of highly abundant proteins is a reasonable and reproducible solution. Often overlooked, clinical tissue specimens also contain a formidable amount of highly abundant blood-derived proteins present in tissue-embedded networks of blood/lymph capillaries and interstitial fluid. Hence, the dynamic range impediment to biomarker discovery remains a formidable obstacle, regardless of clinical sample type (solid tissue and/or body fluid). Thus, we optimized and applied simultaneous immunodepletion of blood-derived proteins from solid tissue and peripheral blood, using clear cell renal cell carcinoma as a model disease. Integrative analysis of data from this approach and genomic data obtained from the same type of tumor revealed concordant key pathways and protein targets germane to clear cell renal cell carcinoma. This includes the activation of the lipogenic pathway characterized by increased expression of adipophilin (PLIN2) along with 'cadherin switching', a phenomenon indicative of transcriptional reprogramming linked to renal epithelial dedifferentiation. We also applied immunodepletion of abundant blood-derived proteins to various tissue types (e.g., adipose tissue and breast tissue) showing unambiguously that the removal of abundant blood-derived proteins represents a powerful tool for the reproducible profiling of tissue proteomes. Herein, we show that the removal of abundant blood-derived proteins from solid tissue specimens is of equal importance to depletion of body fluids and recommend its routine use in the context of biological discovery and/or cancer biomarker research. Finally, this perspective presents the background, rationale and strategy for using tissue-directed high-resolution/accuracy MS-based shotgun proteomics to detect genuine tumor proteins in the peripheral blood of a patient diagnosed with nonmetastatic cancer, employing concurrent liquid chromatography-MS analysis of immunodepleted clinical tissue and blood specimens.

Detection of MicroRNAs in Archival Cytology Urine Smears

MicroRNAs’ dysregulation and profiling have been demonstrated to be clinically relevant in urothelial carcinoma (UC). Urine cytology is commonly used as the mainstay non-invasive test for secondary prevention and follow-up of UC patients. Ancillary tools are needed to support cytopathologists in the diagnosis of low-grade UC. The feasibility and reliability of microRNAs profiling by qRT-PCR analysis (miR-145 and miR-205) in archival routine urine cytology smears (affected by fixation/staining [Papanicolau] and room temperature storage) was tested in a series of 15 non-neoplastic and 10 UC urine specimens. Only samples with >5,000 urothelial cells and with <50% of inflammatory cells/red blood cells clusters were considered. Overall, a satisfactory amount of total RNA was obtained from all the considered samples (mean 1.27±1.43 µg, range 0.06–4.60 µg). Twenty nanograms of total RNA have been calculated to be the minimal total RNA concentration for reliable and reproducible miRNAs expression profiling analysis of archival cytological smears (slope = -3.4084; R-squared = 0.99; efficiency = 1.94). miR-145 and miR-205 were significantly downregulated in UC samples in comparison to non-tumor controls. These findings demonstrate that urine archival cytology smears are suitable for obtaining high-quality RNA to be used in microRNAs expression profiling. Further studies should investigate if miRNAs profiling can be successfully translated into clinical practice as diagnostic or prognostic markers.

Design of recombinant antibody microarrays for urinary proteomics

Urinary proteomics has become a key discipline within clinical proteomics for noninvasive diagnosis and monitoring of disease, and biomarker discovery. In order to decipher complex proteomes, high demands will, however, be placed upon the methodology applied. The purpose of this study was to develop a recombinant antibody microarray platform for urinary proteomics. We adopted our previously in-house developed recombinant antibody microarray set-up and redesigned the platform for urinary proteomics. In this process, the key antibody array assay parameters, such as sample handling, sample labeling protocol, and assay conditions, etc, reflecting the unique properties of urine as sample format, were addressed and reoptimized in a step-by-step procedure. In this proof-of-concept study, we have designed the first generation of a recombinant antibody microarray technology platform for urinary proteomics. The results showed that multiplexed, sensitive (pg/mL range), and reproducible urine protein expression profiling could be performed targeting directly labeled, nonfractionated urine. We have demonstrated that crude, directly labeled urine samples could be profiled in a rapid, reproducible, sensitive, and multiplexed manner after minimal sample prehandling. These findings could potentially pave the way for enhanced urinary proteomics and understanding of renal physiology with implications in both health and disease.

Mass spectrometry of apolipoprotein C-III, a simple analytical method for mucin-type O-glycosylation and its application to an autosomal recessive cutis laxa type-2 (ARCL2) patient

Apolipoprotein C-III (apoCIII) is a small glycoprotein with a single mucin-type core-1 oligosaccharide and is analyzed by isoelectric focusing (IEF) for the diagnosis of genetic defects in O-glycan biosynthesis such as congenital disorders of glycosylation. In the present study, mass spectrometry of apoCIII, after a simple procedure for sample preparation using a small amount of serum, was demonstrated to be a reliable alternative to IEF. It allows reproducible glycan profiling and detection of unglycosylated species. This method was applied to an autosomal recessive cutis laxa type-2 patient and demonstrated decreased site occupancy by O-glycosylation.