Immunoaffinity Enrichment Research Articles

Yeast proteins Gar1p, Nop1p, Npl3p, Nsr1p, and Rps2p are natively methylated and are substrates of the arginine methyltransferase Hmt1p.

The Hmt1 methyltransferase is the predominant arginine methyltransferase in Saccharomyces cerevisiae. There are 18 substrate proteins described for this methyltransferase, however native sites of methylation have only been identified on two of these proteins. Here we used peptide immunoaffinity enrichment, followed by LC-ETD-MS/MS, to discover 21 native sites of arginine methylation on five putative Hmt1 substrate proteins, namely Gar1p (H/ACA ribonucleoprotein complex subunit 1), Nop1p (rRNA 2'-O-methyltransferase fibrillarin), Npl3p (nucleolar protein 3), Nsr1p (nuclear localization sequence-binding protein), and Rps2p (40S ribosomal protein S2). The sites, many of which were found to be mono- or di-methylated, were predominantly found in RGG (Arg-Gly-Gly) motifs. Heavy methyl-SILAC validated the majority of these peptides. The above proteins, and relevant sites of methylation, were subsequently validated by in vitro methylation with recombinant Hmt1. This brings the total of Hmt1 substrate proteins for which native methylation sites have been identified to five.

Identification of Salmonella Typhimurium Deubiquitinase SseL Substrates by Immunoaffinity Enrichment and Quantitative Proteomic Analysis.

Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here, we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification, and global quantitative proteomic analysis. As a model system to identify substrates, we used a virulence-related deubiquitinase, SseL, secreted by Salmonella enterica serovar Typhimurium into host cells. Using this approach, two SseL substrates were identified in the RAW 264.7 murine macrophage-like cell line, S100A6 and heterogeneous nuclear ribonuclear protein K, in addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. This method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.

Abstract 2002: Assembling test points for quantitative proteomics to elucidate RAS signaling

Abstract RAS mutation is a critical player in cancer development, particularly in lung, colon, and pancreas. However, mutant RAS phenotypes have not produced viable therapeutic options. Therefore, additional reagents and techniques are required to study the functionality of RAS pathway components in model systems and patient specimens. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has been applied to study cancer signaling by quantification of protein expression, mutation status, and post-translational modification in a variety of sample types. LC-MRM with immunoaffinity enrichment (ImmunoMRM) enables multiplexed analysis of target peptides with little background interference. Here, we describe the selection of 100 targets for assay development and provide insight into the utility of the immunoMRM platform as part of the NCI's RAS Initiative. Provided with biologically relevant proteins, several approaches were used for peptide selection. Complementary contributions were obtained from analysis of individual proteins, targeted LC-MRM screening, phosphoproteomics, and global discovery proteomics datasets (both in-house and publically available). For phosphorylation and mutation sites, the ability to quantify those molecular endpoints is directly limited by the primary amino acid sequence produced by trypsin digestion. Peptides selected for assay development quantify biological endpoints indicating the expression levels of wild-type and mutant proteins and signaling of RAS pathway components. Literature reviews show the prospective value of each assay in their specific biological context. Assays are developed for RAS signaling proteins in three categories: upstream proteins, RAS isoforms, and downstream effectors. As an example of upstream proteins, EGFR expression and tyrosine phosphorylation will be quantified, which enables comparison of ImmunoMRM to prior techniques. Extensive characterization is planned for RAS isoforms, particularly to monitor the expression of wild-type and mutant proteins. Measurements focused on downstream proteins include Erk and pErk to examine the activation of that MAPK pathway as well as disparate processes, like epithelial-to-mesenchymal transition (N-Cad/E-Cad). A comprehensive circuit diagram is prepared to illustrate the assembled test points for discussion with the cancer biology community. Development of these immunoMRM assays enables evaluation of multiple RAS signaling events and downstream outcomes in one sample. The emerging paradigm of peptide enrichment for protein biomarker measurement provides a novel platform for the study of critical processes in cancer. Although currently early in development, the implementation of this platform can ultimately demonstrate the utility of ImmunoMRM reagent development and be a first step for understanding RAS biology in human tumors and developing novel treatment options for patients. Citation Format: Eric Kuhn, Jeffrey R. Whiteaker, Melissa Martinez, Matthew Holderfield, Jacob Kennedy, Jacob Jaffe, Ping Yan, ChenWei Lin, Gordon Whiteley, Steven A. Carr, Amanda G. Paulovich, John M. Koomen. Assembling test points for quantitative proteomics to elucidate RAS signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2002. doi:10.1158/1538-7445.AM2015-2002

Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex, Quantitative Pharmacodynamic Studies of Phospho-Signaling

In most cell signaling experiments, analytes are measured one Western blot lane at a time in a semiquantitative and often poorly specific manner, limiting our understanding of network biology and hindering the translation of novel therapeutics and diagnostics. We show the feasibility of using multiplex immuno-MRM for phospho-pharmacodynamic measurements, establishing the potential for rapid and precise quantification of cell signaling networks. A 69-plex immuno-MRM assay targeting the DNA damage response network was developed and characterized by response curves and determinations of intra- and inter-assay repeatability. The linear range was ≥ 3 orders of magnitude, the median limit of quantification was 2.0 fmol/mg, the median intra-assay variability was 10% CV, and the median interassay variability was 16% CV. The assay was applied in proof-of-concept studies to immortalized and primary human cells and surgically excised cancer tissues to quantify exposure-response relationships and the effects of a genomic variant (ATM kinase mutation) or pharmacologic (kinase) inhibitor. The study shows the utility of multiplex immuno-MRM for simultaneous quantification of phosphorylated and nonmodified peptides, showing feasibility for development of targeted assay panels to cell signaling networks.

Expanding the yeast protein arginine methylome

Protein arginine methylation is a PTM involved in various cellular processes in eukaryotes. Recent discoveries led to a vast expansion of known sites in higher organisms, indicating that this modification is more widely spread across the proteome than previously assumed. An increased knowledge of sites in lower eukaryotes may facilitate the elucidation of its functions. In this study, we present the discovery of arginine mono-methylation sites in Saccharomyces cerevisiae by a combination of immunoaffinity enrichment and MS/MS. As detection of methylation is prone to yield false positives, we demonstrate the need for stringent measures to avoid elevated false discovery rates. To this end, we employed MethylSILAC in combination with a multistep data analysis strategy. We report 41 unambiguous methylation sites on 13 proteins. Our results indicate that, while substantially less abundant, arginine methylation follows similar patterns as in higher eukaryotes in terms of sequence context and functions of methylated proteins. The majority of sites occur on RNA-binding proteins participating in processes from transcription and splicing to translation and RNA degradation. Additionally, our data suggest a bias for localization of arginine methylation in unstructured regions of proteins, which frequently involves Arg-Gly-Gly motifs or Asn-rich contexts.

Quantitative assessment of human serum transferrin receptor in breast cancer patients pre- and post-chemotherapy using peptide immunoaffinity enrichment coupled with targeted proteomics

BackgroundsTfR, a soluble form of transferrin receptor in serum, has been suggested as an indicator of bone marrow failure in breast cancer patients receiving chemotherapy. However, intensive chemotherapy could also cause a reduction of sTfR to a level below the LOQ of most assays. MethodsAn advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS)–based targeted proteomics assay coupled with peptide immunoaffinity enrichment (SISCAPA) was developed and validated for monitoring sTfR. ResultsTryptic peptide 681VEYHFLSPYVSPK693 was selected as a surrogate analyte for quantification. High-abundant proteins were first removed from serum, followed by SISCAPA that was effective in surrogate peptide enrichment and sensitivity enhancement. The resulting LOQ can achieve 100ng/ml (~10-fold increase). Then, sTfR levels in breast cancer patients pre- and post-chemotherapy, and healthy volunteers were accurately quantified as 1.77±0.53μg/ml, 0.98±0.26μg/ml and 1.66±0.50μg/ml, respectively, using a standard addition method. While there was no evidence for a difference between patients and healthy volunteers, differential levels of sTfR pre- and post-chemotherapy were obtained. Comparison between SISCAPA-targeted proteomics and ELISA indicated that the former approach provided a lower value of sTfR. ConclusionsSISCAPA-targeted proteomics may allow the quantification of low-abundant proteins in a more accurate manner.

Immunoaffinity Enrichment Coupled to Quantitative Mass Spectrometry Reveals Ubiquitin-Mediated Signaling Events

Ubiquitination is one of the most prevalent posttranslational modifications in eukaryotic cells, with functional importance in protein degradation, subcellular localization and signal transduction pathways. Immunoaffinity enrichment coupled with quantitative mass spectrometry enables the in-depth characterization of protein ubiquitination events at the site-specific level. We have applied this strategy to investigate cellular response triggered by two distinct type agents: small molecule inhibitors of the tumor-associated kinases MEK and PI3K or the pro-inflammatory cytokine IL-17. Temporal profiling of protein ubiquitination events across a series of time points covering the biological response permits interrogation of signaling through thousands of quantified proteins, of which only a subset display significant and physiologically meaningful regulation. Distinctive clusters of residues within proteins can display distinct temporal patterns attributable to diverse molecular functions, although the majority of differential ubiquitination appears as a coordinated response across the modifiable residues present within an individual substrate. In cells treated with a combination of MEK and PI3K inhibitors, we found differential ubiquitination of MEK within the first hour after treatment and a series of mitochondria proteins at later time points. In the IL-17 signaling pathway, ubiquitination events on several signaling proteins including HOIL-1 and Tollip were observed. The functional relevance of these putative IL-17 mediators was subsequently validated by knockdown of HOIL-1, HOIP and TOLIP, each of which decreased IL-17-stimulated cytokine production. Together, these data validate proteomic profiling of protein ubiquitination as a viable approach for identifying dynamic signaling components in response to intracellular and extracellular perturbations.

Screening protein isoforms predictive for cancer using immunoaffinity capture and fast LC‐MS in PRM mode

We report an immunocapture strategy to extract proteins known to harbor driver mutations for a defined cancer type before the simultaneous assessment of their mutational status by MS. Such a method bypasses the sensitivity and selectivity issues encountered during the analysis of unfractionated complex biological samples. Fast LC separations using short nanobore columns hyphenated with a high-resolution quadrupole-orbitrap mass spectrometer have been devised to take advantage of fast MS cycle times in conjunction with sharp chromatographic peak widths to accelerate the sample analysis throughput. Such an analytical platform is well suited to analyze simple protein mixtures obtained after immunoaffinity enrichment. After establishing the technical performance of the platform, the method was applied to the quantitative profiling of cellular Ras and EGFR protein isoforms, as well as serum amyloid A isoforms in plasma. Immunoaffinity purification combined with fast LC-MS detection for the detection of driver mutations in tissue and tumor biomarkers in plasma samples can assist clinicians to select an optimal therapeutic intervention for patients.

Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry.

Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system.

Ubiquitination profiling identifies sensitivity factors for IAP antagonist treatment.

Evasion of cell death is one crucial capability acquired by tumour cells to ward-off anti-tumour therapies and represents a fundamental challenge to sustaining clinical efficacy for currently available agents. Inhibitor of apoptosis (IAP) proteins use their ubiquitin E3 ligase activity to promote cancer cell survival by mediating proliferative signalling and blocking cell death in response to diverse stimuli. Using immunoaffinity enrichment and MS, ubiquitination sites on thousands of proteins were profiled upon initiation of cell death by IAP antagonists in IAP antagonist-sensitive and -resistant breast cancer cell lines. Our analyses identified hundreds of proteins with elevated levels of ubiquitin-remnant [K-GG (Lys-Gly-Gly)] peptides upon activation of cell death by the IAP antagonist BV6. The majority of these were observed in BV6-sensitive, but not-resistant, cells. Among these were known pro-apoptotic regulators, including CYC (cytochrome c), RIP1 (receptor-interacting protein 1) and a selection of proteins known to reside in the mitochondria or regulate NF-κB (nuclear factor κB) signalling. Analysis of early time-points revealed that IAP antagonist treatment stimulated rapid ubiquitination of NF-κB signalling proteins, including TRAF2 [TNF (tumour necrosis factor) receptor-associated factor 2], HOIL-1 (haem-oxidized iron-regulatory protein 2 ubiquitin ligase-1), NEMO (NF-κB essential modifier), as well as c-IAP1 (cellular IAP1) auto-ubiquitination. Knockdown of several NF-κB pathway members reduced BV6-induced cell death and TNF production in sensitive cell lines. Importantly, RIP1 was found to be constitutively ubiquitinated in sensitive breast-cancer cell lines at higher basal level than in resistant cell lines. Together, these data show the diverse and temporally defined roles of protein ubiquitination following IAP-antagonist treatment and provide critical insights into predictive diagnostics that may enhance clinical efficacy.

Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA

Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first determination of the success rate (92%) for generating mAbs for immuno-MRM using a recombinant B cell cloning approach, which is considerably faster than the traditional hybridoma approach.

Phosphopeptide immuno-affinity enrichment to enhance detection of tyrosine phosphorylation in plants.

Tyrosine (Tyr) phosphorylation plays an essential role in signaling in animal systems, but the relative contribution of Tyr phosphorylation to plant signal transduction has, until recently, remained an open question. One of the major issues hampering the analysis is the low abundance of Tyr phosphorylation and therefore underrepresentation in most mass spec-based proteomic studies. Here, we describe a working approach to selectively enrich Tyr-phosphorylated peptides from complex plant tissue samples. We describe a detailed protocol that is based on immuno-affinity enrichment step using an anti-phospho-tyrosine (pTyr)-specific antibody. This single enrichment strategy effectively enriches pTyr-containing peptides from complex total plant cell extracts, which can be measured by LC-MS/MS without further fractionation or enrichment.

Sample preparation strategies for targeted proteomics via proteotypic peptides in human blood using liquid chromatography tandem mass spectrometry.

The simultaneous quantification of protein concentrations via proteotypic peptides in human blood by liquid chromatography coupled to quadrupole MS/MS is an important field of bioanalytical research with a high potential for routine diagnostic applications. This review summarizes currently available sample preparation procedures and trends for absolute protein quantification in blood using LC-MS/MS. It discusses approaches of transferring established qualitative protocols to a quantitative analysis regarding their reliability and reproducibility. Techniques used to enhance method sensitivity such as the depletion of high-abundant proteins or the immunoaffinity enrichment of proteins and peptides are described. Furthermore, workflows for (i) protein denaturation, (ii) disulfide bridge reduction and (iii) thiol alkylation as well as (iv) enzymatic digestion for absolute protein quantification are presented. The main focus is on the tryptic digestion as a bottleneck of protein quantification via proteotypic peptides. Conclusively, requirements for a high-throughput application are discussed.

Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis

Nɛ-Acetylation of lysine residues represents a pivotal post-translational modification used by both eukaryotes and prokaryotes to modulate diverse biological processes. Mycobacterium tuberculosis is the causative agent of tuberculosis, one of the most formidable public health threats. Many aspects of the biology of M. tuberculosis remain elusive, in particular the extent and function of Nɛ-lysine acetylation. With a combination of anti-acetyllysine antibody-based immunoaffinity enrichment with high-resolution mass spectrometry, we identified 1128 acetylation sites on 658 acetylated M. tuberculosis proteins. GO analysis of the acetylome showed that acetylated proteins are involved in the regulation of diverse cellular processes including metabolism and protein synthesis. Six types of acetylated peptide sequence motif were revealed from the acetylome. Twenty lysine-acetylated proteins showed homology with acetylated proteins previously identified from Escherichia coli, Salmonella enterica, Bacillus subtilis and Streptomyces roseosporus, with several acetylation sites highly conserved among four or five bacteria, suggesting that acetylated proteins are more conserved. Notably, several proteins including isocitrate lyase involved in the persistence, virulence and antibiotic resistance are acetylated, and site-directed mutagenesis of isocitrate lyase acetylation site to glutamine led to a decrease of the enzyme activity, indicating major roles of KAc in these proteins engaged cellular processes. Our data firstly provides a global survey of M. tuberculosis acetylation, and implicates extensive regulatory role of acetylation in this pathogen. This may serve as an important basis to address the roles of lysine acetylation in M. tuberculosis metabolism, persistence and virulence.

First succinyl-proteome profiling of extensively drug-resistant Mycobacterium tuberculosis revealed involvement of succinylation in cellular physiology.

Protein lysine succinylation, an emerging protein post-translational modification widespread among eukaryotic and prokaryotic cells, represents an important regulator of cellular processes. However, the extent and function of lysine succinylation in Mycobacterium tuberculosis, especially extensively drug-resistant strain, remain elusive. Combining protein/peptide prefractionation, immunoaffinity enrichment, and LC-MS/MS analysis, a total of 686 succinylated proteins and 1739 succinylation sites of M. tuberculosis were identified, representing the first global profiling of M. tuberculosis lysine succinylation. The identified succinylated proteins are involved in a variety of cellular functions such as metabolic processes, transcription, translation, and stress responses and exhibit different subcellular localization via GO, protein interaction network, and other bioinformatic analysis. Notably, proteins involved in protein biosynthesis and carbon metabolism are preferred targets of lysine succinylation. Moreover, two prevalent sequence patterns: EK(suc) and K*****K(suc), can be found around the succinylation sites. There are 109 lysine-succinylated homologues in E. coli, suggesting highly conserved succinylated proteins. Succinylation was found to occur at the active sites predicted by Prosite signature including Rv0946c, indicating that lysine succinylation may affect their activities. There is extensive overlapping between acetylation sites and succinylation sites in M. tuberculosis. Many M. tuberculosis metabolic enzymes and antibiotic resistance proteins were succinylated. This study provides a basis for further characterization of the pathophysiological role of lysine succinylation in M. tuberculosis.

Practical immunoaffinity-enrichment LC-MS for measuring protein kinetics of low-abundance proteins.

For a more complete understanding of pharmacodynamic, metabolic, and pathophysiologic effects, protein kinetics, such as production rate and fractional catabolic rate, can offer substantially more information than protein concentration alone. Kinetic experiments with stable isotope tracers typically require laborious sample preparation and are most often used for studying abundant proteins. Here we describe a practical methodology for measuring isotope enrichment into low-abundance proteins that uses an automated procedure and immunoaffinity enrichment (IA) with LC-MS. Low-abundance plasma proteins cholesteryl ester transfer protein (CETP) and proprotein convertase subtilisin/kexin type 9 (PCSK9) were studied as examples. Human participants (n = 39) were infused with [(2)H(3)]leucine, and blood samples were collected at multiple time points. Sample preparation and analysis were automated and multiplexed to increase throughput. Proteins were concentrated from plasma by use of IA and digested with trypsin to yield proteotypic peptides that were analyzed by microflow chromatography-mass spectrometry to measure isotope enrichment. The IA procedure was optimized to provide the greatest signal intensity. Use of a gel-free method increased throughput while increasing the signal. The intra- and interassay CVs were <15% at all isotope enrichment levels studied. More than 1400 samples were analyzed in <3 weeks without the need for instrument stoppages or user interventions. The use of automated gel-free methods to multiplex the measurement of isotope enrichment was applied to the low-abundance proteins CETP and PCSK9.

Diabetes Induces Lysine Acetylation of Intermediary Metabolism Enzymes in the Kidney

Cells in which insulin is not required for glucose uptake are susceptible to the long-term complications of diabetes. Even in these tissues, however, the major perturbations that would otherwise be engendered by the greatly increased intracellular glucose concentration are mollified by adaptive changes in the enzymes of intermediary metabolism. These include allosteric regulation, product inhibition, and covalent modification as well as alterations in gene transcription. More recently, advances in proteomic technology have shown that reversible acetylation of the ε-amino group of lysine provides an additional means of modulating protein function and, in particular, enzyme activity. Here, we explored the extent of protein acetylation in an organ susceptible to the long-term complications of diabetes, examining the kidneys of rats with streptozotocin-induced diabetes and kidney cells exposed to high glucose. Using high-resolution mass spectrometry coupled with immunoaffinity enrichment, we identified 47 lysine-acetylated proteins in the kidneys of diabetic rats compared with 11 in control kidneys. Bioinformatic interrogation of the acetylome from diabetic animals showed a predominance of metabolic pathway involvement including the citrate acid cycle, glycolysis/gluconeogenesis, and metabolism of branched chain amino acids. Increased lysine acetylation was also noted in mesangial and tubular cells exposed to 25 mmol/L compared with 5.6 mmol/L glucose. These findings highlight acetylation as a posttranslational modification affecting numerous proteins. Current drug discovery efforts to develop small molecule inhibitors and activators of various lysine acetylases and deacetylases offer a new potential strategy to reduce the likelihood of diabetes complications.

Targeted quantification of C-reactive protein and cystatin c and its variants by immuno-MALDI-MS.

The most common technologies for quantitative determination of protein biomarkers are immunoassays, which exist in various formats. Immunoassays offer sensitive and fast protein quantification, but can hardly discriminate between protein variants. Post-translational modifications and genetic variants increase protein microheterogeneity and may play important roles in biological processes. Mass spectrometry combined with immunoaffinity enrichment detects protein microheterogeneity and can quantify different isoforms. We here present an immuno-MALDI-MS approach for the combined quantification of two important biomarkers of inflammation and renal function, C-reactive protein (CRP) and cystatin C, respectively. Antibodies were immobilized onto reversed-phase tips, which allows easy and flexible sample processing. Quantification was performed in singleplex and duplex assays, and characteristics were evaluated for different internal standards, i.e., PEGylated and polyhistidine-tagged proteins. The best performances were obtained for polyhistidine-tagged standards with respect to limits of detection (CRP, 0.10 μg/mL; cystatin C, 0.003 μg/mL) and coefficients of variation (CRP, 2.4-7.0%; cystatin C, 3.0-8.9%). The methods were benchmarked against immunoturbidimetry and nephelometry and demonstrated good between-assay agreement (R(2) = 0.989 for CRP; R(2) = 0.939 for cystatin C). Several variants of cystatin C were identified and quantified, while none were observed for CRP. This immuno-MALDI method describes a novel approach for targeted quantitative investigation of protein microheterogeneity and is well suited for assessment of biomarker status in precious samples from biobanks due to its low sample consumption.

Quantification of Tau in Cerebrospinal Fluid by Immunoaffinity Enrichment and Tandem Mass Spectrometry

Cerebrospinal fluid (CSF) tau is a common biomarker for Alzheimer disease (AD). Measurements of tau have historically been performed using immunoassays. Given the molecular diversity of tau in CSF, the selectivity of these immunoassays has often been questioned. Therefore, we aimed to develop an analytically sensitive and selective immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) (IA-MS) assay. IA-MS sample analysis involved the addition of an internal standard, immunoaffinity purification of tau using a tau monoclonal antibody coupled to magnetic beads, trypsin digestion, and quantification of a surrogate tau peptide by LC-MS/MS using a Waters Trizaic nanoTile ultraperformance LC microfluidic device. Further characterization of tau peptides was performed by full-scan MS using a Thermo Orbitrap LC-MS. CSF samples from a cohort of age-matched controls and patients with AD were analyzed by the IA-MS method as well as a commercially available immunoassay. The IA-MS assay had intra- and interassay imprecision values of 3.2% to 8.1% CV and 7.8% to 18.9% C, respectively, a mean recovery of 106%, and a limit of quantification of 0.25 pmol/L and was able to quantify tau concentrations in all human specimens tested. The IA-MS assay showed a correlation of R(2) = 0.950 against a total-tau immunoassay. In patients with AD, tau was increased approximately 2-fold. Combining immunoaffinity enrichment with microflow LC-MS/MS analysis is an effective approach for the development of a highly selective assay to measure total tau and, potentially, other posttranslationally modified forms of tau in CSF.

Investigational new insulin glargine 300 U/ml has the same metabolism as insulin glargine 100 U/ml

Insulin glargine is processed in vivo into soluble 21A-Gly-human insulin (M1), the principal moiety responsible for metabolic effects, and subsequently into M2. This sub-study compared metabolism and metabolite pharmacokinetic (PK) profiles of investigational new insulin glargine U300 (Gla-300) with insulin glargine 100 U/ml (Gla-100, Lantus®, Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany) in people with type 1 diabetes. Participants received 0.4 (n = 18) or 0.6 U/kg Gla-300 (n = 12), and 0.4 U/kg Gla-100 (n = 30) once daily in randomized order for 8 days prior to a 36-h euglycaemic clamp. Metabolites were quantified using immunoaffinity enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS). Glargine metabolism was the same regardless of Gla-100 or Gla-300 administration; M1 was confirmed as the principal active moiety circulating in blood. Steady state concentrations of M1 were achieved after 2 days for Gla-100, and 4 days for Gla-300. Steady state M1 values defined prolonged and even flatter PK profiles after Gla-300 administration compared with M1 profiles after Gla-100.