Targeting Interferon Alpha Subtypes in Serum: A Comparison of Analytical Approaches to the Detection and Quantitation of Proteins in Complex Biological Matrices

The targeted detection and quantitation of proteins in complex biological fluids such as blood is as analytically challenging as it is crucial for biomedical research. Antibody-based techniques such as the ELISA are the current standards for such measurements, having in favorable cases high specificity and pg/mL detection limits. Long development timelines and susceptibility to cross reactivity have led researchers to investigate mass spectrometric alternatives. The literature contains diverse schemes for sample preparation and multiple platforms for mass spectrometric detection. Critical evaluations of competing technologies are, however, badly needed. Taking closely related subtypes of the pro-inflammatory cytokine interferon alpha as a test case, we compared a sample preparation workflow based on affinity enrichment to one based on generic multidimensional chromatography, and evaluated mass spectrometric techniques using tandem mass spectrometry on low resolution ion traps, high resolution “accurate mass tags,” and triple quadrupole selective reaction monitoring. Each workflow and detection method proved capable of detecting and discriminating between these proteins at or below the ng/mL level in human serum. Quantitation by isotope dilution was evaluated using full length protein as the internal standard. Both triple quadrupole selected reaction monitoring and orbitrap selected ion monitoring produced linear calibration curves from 1 ng/mL to 1 μg/mL, with lower limits of quantitation below 5 and 50 ng/mL, respectively

Targeting Interferon Alpha Subtypes in Serum: A Comparison of Analytical Approaches to the Detection and Quantitation of Proteins in Complex Biological Matrices

The targeted detection and quantitation of proteins in complex biological fluids such as blood is as analytically challenging as it is crucial for biomedical research. Antibody-based techniques such as the ELISA are the current standards for such measurements, having in favorable cases high specificity and pg/mL detection limits. Long development timelines and susceptibility to cross reactivity have led researchers to investigate mass spectrometric alternatives. The literature contains diverse schemes for sample preparation and multiple platforms for mass spectrometric detection. Critical evaluations of competing technologies are, however, badly needed. Taking closely related subtypes of the pro-inflammatory cytokine interferon alpha as a test case, we compared a sample preparation workflow based on affinity enrichment to one based on generic multidimensional chromatography, and evaluated mass spectrometric techniques using tandem mass spectrometry on low resolution ion traps, high resolution “accurate mass tags,” and triple quadrupole selective reaction monitoring. Each workflow and detection method proved capable of detecting and discriminating between these proteins at or below the ng/mL level in human serum. Quantitation by isotope dilution was evaluated using full length protein as the internal standard. Both triple quadrupole selected reaction monitoring and orbitrap selected ion monitoring produced linear calibration curves from 1 ng/mL to 1 μg/mL, with lower limits of quantitation below 5 and 50 ng/mL, respectively

The transcription factor ATF4 binds and regulates the <i>NLRP1</i> gene promoter.

<p>(A) Different <i>NLRP1</i> promoter regions were cloned into a luciferase reporter vector. At 24 hours after transfection, HeLa cells were treated with BFA for 20 hours before measuring luciferase activity. (B) HeLa cells were transfected with wild-type <i>NLRP1</i> promoter or a version carrying mutations in the ATF4-binding motif. At 24 hours after transfection, cells were treated with BFA for 20 hours before measuring luciferase activity. (C) The indicated <i>NLRP1</i> promoter-luciferase vectors were transfected into either wild-type or <i>ATF4</i>^{<i>−/−</i>} HeLa cells. At 24 hours after transfection, cells were treated with BFA for 20 hours before measuring luciferase activity. (D) ChIP analysis of un-stimulated (DMSO) or BFA-stimulated HeLa cells, followed by qPCR analysis of ATF4 occupancy at the NLRP1 and ATF3 promoter. ATF3 was used as positive control. Each panel is representative of at least three independent experiments.</p

A Turn-Key Approach for Large-Scale Identification of Complex Posttranslational Modifications

The conjugation of complex post-translational modifications (PTMs) such as glycosylation and Small Ubiquitin-like Modification (SUMOylation) to a substrate protein can substantially change the resulting peptide fragmentation pattern compared to its unmodified counterpart, making current database search methods inappropriate for the identification of tandem mass (MS/MS) spectra from such modified peptides. Traditionally it has been difficult to develop new algorithms to identify these atypical peptides because of the lack of a large set of annotated spectra from which to learn the altered fragmentation pattern. Using SUMOylation as an example, we propose a novel approach to generate large MS/MS training data from modified peptides and derive an algorithm that learns properties of PTM-specific fragmentation from such training data. Benchmark tests on data sets of varying complexity show that our method is 80–300% more sensitive than current state-of-the-art approaches. The core concepts of our method are readily applicable to developing algorithms for the identifications of peptides with other complex PTMs

Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

<div><p>Perturbation of endoplasmic reticulum (ER) homeostasis triggers the ER stress response (also known as Unfolded Protein Response), a hallmark of many pathological disorders. However the connection between ER stress and inflammation remains largely unexplored. Recent data suggest that ER stress controls the activity of inflammasomes, key signaling platforms that mediate innate immune responses. Here we report that expression of NLRP1, a core inflammasome component, is specifically up-regulated during severe ER stress conditions in human cell lines. Both IRE1α and PERK, but not the ATF6 pathway, modulate <i>NLRP1</i> gene expression. Furthermore, using mutagenesis, chromatin immunoprecipitation and CRISPR-Cas9-mediated genome editing technology, we demonstrate that ATF4 transcription factor directly binds to <i>NLRP1</i> promoter during ER stress. Although involved in different types of inflammatory responses, XBP-1 splicing was not required for <i>NLRP1</i> induction. This study provides further evidence that links ER stress with innate</p></div

Atf4 but not Xbp-1s stimulates <i>NLRP1</i> gene expression during ER stress.

<p>(A) HeLa cells were infected with increasing concentrations of murine Xbp-1s and Atf4 adenovirus for 24 hours and NLRP1 mRNA was measured by qPCR. (B) IRE1α, PERK, ATF6 and XBP-1s were down-regulated using siRNA in HeLa cells. Cells were treated with BFA for 20 hours and mRNA levels were measured by qPCR. IRE1α, PERK, ATF6 and XBP-1s knock-down was verified by SDS-PAGE/immunoblotting. Each panel is representative of at least three independent experiments.</p

NLRP1 mRNA and protein are up-regulated upon ER stress.

<p>(A) Un-differentiated THP-1 cells were treated with the indicated stimuli for 6 hours. NLRP1 levels were measured by quantitative real-time PCR (qPCR) using cyclophillin A as an endogenous control. Semi-quantitative RT-PCR using a different NLRP1 primer set and GAPDH as a control is also shown. (B) HeLa cells were treated either with BFA or TG for the indicated times. NLRP1 mRNA levels were measured by qPCR and RT-PCR. Spliced and un-spliced XBP-1 forms were also evaluated by RT-PCR. (C) HCT116 cells were treated with the indicated stimuli for 24 hours. NLRP1 and NOD1 mRNA levels were measured by qPCR. (D) Cell lysates from wild-type or <i>NLRP1</i>^{<i>−/−</i>} HeLa, THP-1 and K562 cells, untreated or treated with BFA for 20 hours, were normalized for total protein content. Cell extracts were then subjected to SDS-PAGE/immunoblot analysis before and after immunoprecipitation with NLRP1 antibody. Vinculin was detected as loading control. NLRP1 mRNA levels were also measured by RT-PCR. Each panel is representative of at least three independent experiments. (DMSO: dimethyl sulfoxide, TM: tunicamycin, TG: thapsigargin, MSU: monosodium urate crystals, BFA: brefeldin A, PolyI:C: polyinosinic-polycytidylic acid, FLA: flagellin, MDP: muramyl dipeptide, R837: Imiquimod)</p

NLRP1 mRNA up-regulation is dependent on both IRE1α and PERK pathways.

<p>(A) IRE1α, PERK and ATF6 levels were reduced using siRNA. Upon treatment with ER stress, mRNA levels were measured by qPCR and RT-PCR. IRE1α, PERK and ATF6 knock-down was verified by SDS-PAGE/immunoblotting. (B) Stably transduced HeLa cells were cultured in presence or absence of doxycycline (Dox) for 24 hours and then treated overnight with 2μM BFA. mRNA levels were measured by qPCR and RT-PCR. IRE1α, PERK and ATF6 knock-down was verified by SDS-PAGE/immunoblotting. Each panel is representative of at least three independent experiments.</p

Discovery and Qualification of Candidate Urinary Biomarkers of Disease Activity in Lupus Nephritis

Lupus nephritis (LN) is a severe clinical manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity and mortality. Assessment of severity and activity of renal involvement in SLE requires a kidney biopsy, an invasive procedure with limited prognostic value. Noninvasive biomarkers are needed to inform treatment decisions and to monitor disease activity. Proteinuria is associated with disease progression in LN; however, the composition of the LN urinary proteome remains incompletely characterized. To address this, we profiled LN urine samples using complementary mass spectrometry-based methods:  protein gel fractionation, chemical labeling using tandem mass tags, and data-independent acquisition. Combining results from these approaches yielded quantitative information on 2573 unique proteins in urine from LN patients. A multiple-reaction monitoring (MRM) method was established to confirm eight proteins in an independent cohort of LN patients, and seven proteins (transferrin, α-2-macroglobulin, haptoglobin, afamin, α-1-antitrypsin, vimentin, and ceruloplasmin) were confirmed to be elevated in LN urine compared to healthy controls. In this study, we demonstrate that deep mass spectrometry profiling of a small number of patient samples can identify high-quality biomarkers that replicate in an independent LN disease cohort. These biomarkers are being used to inform clinical biomarker strategies to support longitudinal and interventional studies focused on evaluating disease progression and treatment efficacy of novel LN therapeutics

Discovery and Qualification of Candidate Urinary Biomarkers of Disease Activity in Lupus Nephritis

Lupus nephritis (LN) is a severe clinical manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity and mortality. Assessment of severity and activity of renal involvement in SLE requires a kidney biopsy, an invasive procedure with limited prognostic value. Noninvasive biomarkers are needed to inform treatment decisions and to monitor disease activity. Proteinuria is associated with disease progression in LN; however, the composition of the LN urinary proteome remains incompletely characterized. To address this, we profiled LN urine samples using complementary mass spectrometry-based methods:  protein gel fractionation, chemical labeling using tandem mass tags, and data-independent acquisition. Combining results from these approaches yielded quantitative information on 2573 unique proteins in urine from LN patients. A multiple-reaction monitoring (MRM) method was established to confirm eight proteins in an independent cohort of LN patients, and seven proteins (transferrin, α-2-macroglobulin, haptoglobin, afamin, α-1-antitrypsin, vimentin, and ceruloplasmin) were confirmed to be elevated in LN urine compared to healthy controls. In this study, we demonstrate that deep mass spectrometry profiling of a small number of patient samples can identify high-quality biomarkers that replicate in an independent LN disease cohort. These biomarkers are being used to inform clinical biomarker strategies to support longitudinal and interventional studies focused on evaluating disease progression and treatment efficacy of novel LN therapeutics