Shotgun glycomics: a microarray strategy for functional glycomics.

Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a class of glycoconjugates that is challenging to study, recognized by toxins, antibodies and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. We separated fluorescent GSLs by multidimensional chromatography, quantified them and coupled them to glass slides to create GSL shotgun microarrays. Then we interrogated the microarrays with cholera toxin, antibodies and sera from individuals with Lyme disease to identify biologically relevant GSLs that we subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans is an approach for accessing the complex glycomes of animal cells and is a strategy for focusing structural analyses on functionally important glycans.

Utilization of assay performance characteristics to estimate hemoglobin A1c result reliability.

BACKGROUND: Allowable total error (TE(a)) goals for hemoglobin (Hb) A(1c) require minimal assay imprecision and bias and implementation of a robust QC monitoring program. Here, we compare the combined influence on the risk of reporting unreliable results of TE(a) goals, a routine QC practice, and assay performance characteristics of 6 Hb A(1c) instruments across 4 academic medical centers. METHODS: The CLSI protocols EP-5 and EP-9 were applied to investigate Hb A(1c) result imprecision and bias on the Variant II Turbo and Variant II (Bio-Rad), G8 (Tosoh), Capillarys 2 Flex Piercing (Sebia), COBAS Integra 800 (Roche), and DCA Vantage (Siemens). Patient-weighted σ values and the risk of reporting unreliable Hb A(1c) results were determined for each assay at TE(a) specifications of 5%, 6%, and 7%. RESULTS: A large range of patient-weighted σ values spanning 0.5 orders of magnitude at a 6% TE(a) was observed. Although imprecision for all instruments was <3%, bias impacted the majority of the σ changes observed. Estimates for reporting unreliable results varied almost 500-fold based on analytical performance alone. CONCLUSIONS: Considerable differences in the probability of reporting unreliable Hb A(1c) results between different NGSP (formerly the National Glycohemoglobin Standardization Program)-certified platforms were observed. At a 6% TE(a), our study indicates all but the Capillarys 2 Flex Piercing requires that the maximum affordable QC be run. Risk estimates for individual laboratories' Hb A(1c) methods can be used to assess QC practices and residual risk of an unreliable Hb A(1c) result.

The proprotein convertase encoded by amontillado (amon) is required in Drosophila corpora cardiaca endocrine cells producing the glucose regulatory hormone AKH.

Peptide hormones are potent signaling molecules that coordinate animal physiology, behavior, and development. A key step in activation of these peptide signals is their proteolytic processing from propeptide precursors by a family of proteases, the subtilisin-like proprotein convertases (PCs). Here, we report the functional dissection of amontillado (amon), which encodes the Drosophila homolog of the mammalian PC2 protein, using cell-type specific inactivation and rescue experiments, and we show that amon is required in the islet-like adipokinetic hormone (AKH)-producing cells that regulate sugar homeostasis. In Drosophila, AKH acts analogously to vertebrate glucagon to increase circulating sugar levels from energy stores, while insulin-like peptides (DILPs) act to decrease sugar levels. amon mutant larvae have significantly reduced hemolymph sugar levels, and thus phenocopy larvae where the AKH-producing cells in the corpora cardiaca have been ablated. Reduction of amon expression in these cells via cell-specific RNA inactivation also results in larvae with reduced sugar levels while expression of amon in AKH cells in an amon mutant background rescues hypoglycemia. Hypoglycemia in larvae resulting from amon RNA inactivation in the AKH cells can be rescued by global expression of the akh gene. Finally, mass spectrometric profiling shows that the production of mature AKH is inhibited in amon mutants. Our data indicate that amon function in the AKH cells is necessary to maintain normal sugar homeostasis, that amon functions upstream of akh, and that loss of mature AKH is correlated with loss of amon activity. These observations indicate that the AKH propeptide is a proteolytic target of the amon proprotein convertase and provide evidence for a conserved role of PC2 in processing metabolic peptide hormones.

Deficiency of prohormone convertase dPC2 (AMONTILLADO) results in impaired production of bioactive neuropeptide hormones in Drosophila.

Peptide hormones synthesized by secretory neurons in the CNS are important regulators of physiology, behavior, and development. Like other neuropeptides, they are synthesized from larger precursor molecules by a specific set of enzymes. Using a combination of neurogenetics, immunostainings, and direct mass spectrometric profiling, we show that the presence of Drosophila prohormone convertase 2 encoded by the gene amontillado (amon) is a prerequisite for the proper processing of neuropeptide hormones from the major neurohemal organs of the CNS. A loss of amon correlates with a loss of neuropeptide hormone signals from the larval ring gland and perisympathetic organs. Neuropeptide hormone signals were still detectable in the adult corpora cardiaca of older amon-deficient flies which were amon heat-shock-rescued until eclosion. A semiquantification by direct peptide profiling using stable isotopic standards showed, however, that their neuropeptide hormone levels are strongly reduced. Targeted expression of GFP under the control of amon regulatory regions revealed a co-localization with the investigated peptide hormones in secretory neurons of the brain and ventral nerve cord. The lack of AMON activity resulted in a deficiency of L3 larva to enter the wandering phase. In conclusion, our findings provide the first direct evidence that AMON is a key enzyme in the production of neuropeptides in the fruitfly.

Cancer biomarkers: surviving the journey from bench to bedside.

In summary, the abundance of reported candidate-biomarker proteins in the scientific literature compared to the lack of those reaching clinical use indicates that the aforementioned pipeline bottleneck falls in either the verification or validation phases. To stress this point, Polanski and Anderson compiled a list of 1,261 proteins that have been cited in the literature as being differentially expressed in human cancers.¹ Of the 1,261 proteins, 22% are reported to be present in the blood and should be detectable given a sensitive enough assay. Interestingly, only 5% of these candidates have been thoroughly investigated as biomarkers (greater than 500 citations),¹ with 41 (~3%) actually being used in some clinical capacity. The reason behind so few biomarkers reaching the clinic can largely be explained by the inability of current technologies to consistently and quantitatively verify the presence of the candidates in patient samples and the failure, thus far, to identify biomarkers with high specificity for a particular disease.9 As noted above, none of the nine FDA-approved cancer biomarkers demonstrate the specificity required for diagnosis when used alone. Thus, the development of panels of proteins, such as the FDA-approved OVA1 test,57 may be crucial to achieve the specificity required for early cancer diagnosis, and is interesting to speculate that members of such panels are likely to have already been identified but not yet implemented.58

Disruption of the A-kinase anchoring domain in flagellar radial spoke protein 3 results in unregulated axonemal cAMP-dependent protein kinase activity and abnormal flagellar motility.

Biochemical studies of Chlamydomonas flagellar axonemes revealed that radial spoke protein (RSP) 3 is an A-kinase anchoring protein (AKAP). To determine the physiological role of PKA anchoring in the axoneme, an RSP3 mutant, pf14, was transformed with an RSP3 gene containing a mutation in the PKA-binding domain. Analysis of several independent transformants revealed that the transformed cells exhibit an unusual phenotype: a fraction of the cells swim normally; the remainder of the cells twitch feebly or are paralyzed. The abnormal/paralyzed motility is not due to an obvious deficiency of radial spoke assembly, and the phenotype cosegregates with the mutant RSP3. We postulated that paralysis was due to failure in targeting and regulation of axonemal cAMP-dependent protein kinase (PKA). To test this, reactivation experiments of demembranated cells were performed in the absence or presence of PKA inhibitors. Importantly, motility in reactivated cell models mimicked the live cell phenotype with nearly equal fractions of motile and paralyzed cells. PKA inhibitors resulted in a twofold increase in the number of motile cells, rescuing paralysis. These results confirm that flagellar RSP3 is an AKAP and reveal that a mutation in the PKA binding domain results in unregulated axonemal PKA activity and inhibition of normal motility.

Pathology consultation on HbA(1c) methods and interferences.

OBJECTIVES: To review the various hemoglobin (Hb) A1c methods, with a focus on interferences resulting from Hb variants. METHODS: HbA1c is a marker used for the diagnosis and management of diabetes. Each available HbA1c method has advantages and challenges, such as its susceptibility to interferences. RESULTS: The presence of Hb variants and/or abnormalities in RBC turnover cannot only interfere analytically with HbA1c measurements but also may affect the clinical interpretation of HbA1c values. CONCLUSIONS: Familiarity with the advantages and challenges of the various methods used for HbA1c testing is essential when establishing therapeutic management and goals based on HbA1c results, especially in populations with a high prevalence of Hb variants.

Challenges in implementing clinical liquid chromatography-tandem mass spectrometry methods--the light at the end of the tunnel.

The use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the clinical setting is a relatively new application. One of the significant barriers hampering the transition of LC-MS/MS from the research lab into a clinical setting is the uncertainty of how to successfully develop and validate a method that meets guidelines for clinical applications. Here, we have taken this seemingly overwhelming process and broken it into five general stages for consideration: assessing the clinical validity of a new LC-MS/MS assay, determination of feasibility, assay development, assay validation and post-implementation monitoring. Although various publications are available and serve as resources for determining development processes and acceptability criteria for specific LC-MS/MS assays, many of them are general recommendations or are specific to research applications that may not translate either practically or clinically. In this perspective special feature article, a resource is compiled that describes key differences between LC-MS/MS methods for research use versus clinical use. In addition, the challenges facing the expanding role of this technique in the clinical setting are discussed, including instrumentation/automation challenges, potential regulation of laboratory developed tests by the US Food and Drug Administration and standardization and harmonization of MS methods through the use of traceable materials and availability of guidance documents.

Direct total and free testosterone measurement by liquid chromatography tandem mass spectrometry across two different platforms.

OBJECTIVES: To develop and validate liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for the direct measurement of total and free testosterone in patient samples on two different analytical systems. DESIGN AND METHODS: An API 4000 and 5000 triple quadropoles were used and compared; the former is reported to be 3-5 times less sensitive, as was used to set the quantitation limits. Free testosterone was separated from the protein-bound fraction by equilibrium dialysis followed by derivatization. Either free or total testosterone, and a deuterated internal standard (d3-testosterone) were extracted by liquid-liquid extraction. The validation results were compared to two different clinical laboratories. RESULTS: The use of d2-testosterone was found to be unacceptable for our method. The total testosterone LC-MS/MS methods on both systems were linear over a wide concentration range of 1.5-2000ng/dL. Free testosterone was measured directly using equilibrium dialysis coupled LC-MS/MS and linear over the concentration range of 2.5-2500pg/mL. Good correlation (total testosterone, R(2)=0.96; free testosterone, R(2)=0.98) was observed between our LC-MS/MS systems and comparator laboratory. However, differences in absolute values for both free and total testosterone measurements were observed while a comparison to a second published LC-MS/MS method showed excellent correlation. Free and total testosterone measurements correlated well with clinical observations. CONCLUSIONS: To our knowledge, this is the first published validation of free and total testosterone methods across two analytical systems of different analytical sensitivities. A less sensitive system does not sacrifice analytical or clinical sensitivity to directly measure free and total testosterone in patient samples.

Development of a liquid chromatography tandem mass spectrometry method for iothalamate measurement to assess renal function for potential kidney donation.

BACKGROUND: Chronic kidney disease often goes undetected due to the insensitivity of current methods to accurately assess glomerular filtration rate (GFR) in early stages of renal dysfunction. The clearance of exogenously introduced iothalamate, a commonly used radiopaque agent, is an alternative to inulin clearance for the assessment of renal function and its use in calculating GFR can serve as a screening tool for kidney transplant donors. METHODS: A method was developed to measure iothalamate in plasma and urine samples by HPLC combined with electrospray positive ionization tandem mass spectrometry (MS/MS). Iothalamate is isolated from plasma by methanol extraction and urine using a quick-spin filtration approach, then monitored by multiple reaction monitoring using the hydrogen adduct mass transitions. Iohexol was used as an internal standard. RESULTS: Iothalamate was measured within an analytical run time of 5 min, with a lower limit of quantification of 18.75 ng/ml. The intraassay and interassay variations of the plasma and urine iothalamate assays were both <9%. Recovery from plasma and urine samples ranged from 93.6% to 104.1%. GFR was calculated using the patient's urine flow rate and plasma and urine iothalamate values. Linear correlations tested by LC-MS/MS and an accepted capillary electrophoresis (CE) assay showed similar results (GFR, r=0.92, Sy/x=10.3). CONCLUSIONS: We developed and validated an LC-MS/MS method for quantitating iothalamate in plasma and urine to calculate GFR used for screening potential kidney donors in our hospital system. A less sensitive mass spectrometry system does not sacrifice analytical or clinical sensitivity for measuring GFR.

Unintended reporting of misleading Hb A(1c) values when using assays incapable of detecting hemoglobin variants.

CONTEXT: It is recommended that hemoglobin (Hb) A1c (Hb A1c) not be used to assess average glycemia in patients who have altered red blood cell life span. OBJECTIVE: To investigate the frequency of reporting an Hb A1c value for Hb variant samples that do not include Hb A. DESIGN: Hb A1c samples (n = 500) were procured and screened for Hb variants that may affect Hb A1c interpretation (Hb SS, Hb SC, and Hb S-ß-thalassemia). Five of each of these samples were tested by ion-exchange high-performance liquid chromatography, immunoturbidimetric assay, second-generation immunoturbidimetric assay, and affinity chromatography. RESULTS: Eleven (2.2%) homozygous Hb SS, 6 (1.2%) Hb SC, and 5 (1.0%) Hb S-ß-thalassemia samples were identified out of the 500 samples tested. Three of 4 instruments investigated in this study are known to not be plagued by analytic interference from these Hb variants but disturbingly reported Hb A1c values in the absence of Hb A. CONCLUSIONS: The improved analytic specificity of Hb A1c platforms has by and large eliminated interferences from the most common heterozygous Hb variants. A consequence, however, is the potential for unintended reporting of Hb A1c results in the presence of homozygous and compound heterozygous Hb variants that lack Hb A and the inability to distinguish those samples not recommended to be used for patient care. The ability to identify samples harboring Hb variants that preclude the utility of Hb A1c may be beneficial in high prevalence populations.

Development of a fast and simple liquid chromatography-tandem mass spectrometry method for the quantitation of argatroban in patient plasma samples.

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the direct measurement of argatroban in human plasma was developed and compared with the activity-based Hemoclot Thrombin Inhibitors assay. UPLC-MS/MS was performed using diclofenac as an internal standard. In summary, argatroban and diclofenac were extracted from 100 µL of plasma using a methanol precipitation protocol, and chromatographic separation was performed on an ACQUITY TQD mass spectrometer using a UPLC C18 BEH 1.7 µm column with a water and methanol gradient containing 0.1% formic acid. The detection and quantitation were performed using positive ion electrospray ionization and multiple reaction monitoring (MRM) mode. The UPLC-MS/MS method was linear over the concentration range of 0.003-3.0 µg/mL, with a lower limit of quantitation for argatroban of 0.003 µg/mL. The intra- and inter-assay imprecision was less than 12% at the plasma argatroban concentrations tested. Good correlation was demonstrated between the UPLC-MS/MS method and the indirect activity-based assay for determination of argatroban. However, increased plasma fibrinogen levels caused underestimation of argatroban levels using the indirect activity-based assay, whereas the UPLC-MS/MS method was unaffected. UPLC-MS/MS provides a relatively simple, sensitive, and rapid means of argatroban monitoring. It has successfully been applied to assess plasma argatroban concentrations in hospitalized patients and may provide a more accurate determination of argatroban concentrations than an activity-based assay in certain clinical conditions.

Plasma and cerebral spinal fluid tranexamic acid quantitation in cardiopulmonary bypass patients.

A method for the determination of tranexamic acid (TXA) in human plasma and cerebral spinal fluid (CSF) was developed. Analyses were performed by ultra performance liquid chromatography with tandem mass spectrometry detection (UPLC-MS/MS) using ɛ-aminocaproic acid (ACA) as an internal standard. TXA and ACA were extracted from a 50 µL sample of plasma or CSF using a methanol protein crash protocol, and chromatographic separation was performed on an ACQUITY™ TQD mass spectrometer using a UPLC C18 BEH 1.7 µm column with a water and methanol gradient containing 0.1% formic acid. The detection and quantitation was performed by positive ion electrospray ionization using the multiple reaction monitoring (MRM) mode. The method was linear over the concentration range of 0.1-10.0 µg/mL, with lower limit of quantitation of 0.1 µg/mL for TXA. The intra- and inter-assay precision was less than 12% and 13% respectively at the plasma and CSF TXA concentrations tested. The present method provides a relatively simple and sensitive assay with short turn-around-time. The method has been successfully applied to assess the plasma and CSF concentrations of tranexamic acid achieved with only one dosing regimen of tranexamic acid in patients undergoing cardiopulmonary bypass surgery (CPB).

Antibody responses against xenotropic murine leukemia virus-related virus envelope in a murine model.

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) was recently discovered to be the first human gammaretrovirus that is associated with chronic fatigue syndrome and prostate cancer (PC). Although a mechanism for XMRV carcinogenesis is yet to be established, this virus belongs to the family of gammaretroviruses well known for their ability to induce cancer in the infected hosts. Since its original identification XMRV has been detected in several independent investigations; however, at this time significant controversy remains regarding reports of XMRV detection/prevalence in other cohorts and cell type/tissue distribution. The potential risk of human infection, coupled with the lack of knowledge about the basic biology of XMRV, warrants further research, including investigation of adaptive immune responses. To study immunogenicity in vivo, we vaccinated mice with a combination of recombinant vectors expressing codon-optimized sequences of XMRV gag and env genes and virus-like particles (VLP) that had the size and morphology of live infectious XMRV. RESULTS: Immunization elicited Env-specific binding and neutralizing antibodies (NAb) against XMRV in mice. The peak titers for ELISA-binding antibodies and NAb were 1:1024 and 1:464, respectively; however, high ELISA-binding and NAb titers were not sustained and persisted for less than three weeks after immunizations. CONCLUSIONS: Vaccine-induced XMRV Env antibody titers were transiently high, but their duration was short. The relatively rapid diminution in antibody levels may in part explain the differing prevalences reported for XMRV in various prostate cancer and chronic fatigue syndrome cohorts. The low level of immunogenicity observed in the present study may be characteristic of a natural XMRV infection in humans.

Mass spectrometry-coupled techniques for viral-related disease biomarker identification.

The advent of high-resolution mass spectrometers coupled with proteomic techniques has facilitated the discovery and characterization of novel viral proteins and the detection of virus-induced changes in the cellular proteome. These advances have enabled a more comprehensive characterization of viral interactions involved in infection and pathogenesis, and allowed the discovery of viral biomarkers. This article focuses on the role of mass spectrometry proteomic techniques to identify and characterize both prospective and verified viral biomarkers, and their implications on the diagnosis of disease.