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In JoVE (1)
Other Publications (19)
- Behaviour Research and Therapy
- Journal of Traumatic Stress
- Behaviour Research and Therapy
- Behaviour Research and Therapy
- Proteomics
- Behaviour Research and Therapy
- Arthritis and Rheumatism
- Assay and Drug Development Technologies
- Behaviour Research and Therapy
- Journal of Traumatic Stress
- Behaviour Research and Therapy
- Molecular & Cellular Proteomics : MCP
- Journal of Psychosomatic Research
- Psychiatric Services (Washington, D.C.)
- Clinical Chemistry
- Molecular & Cellular Proteomics : MCP
- Journal of Traumatic Stress
- Molecular & Cellular Proteomics : MCP
- Molecular & Cellular Proteomics : MCP
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Articles by Eric Kuhn in JoVE
JoVE General
Proteinler Peptid immunoaffiniti Zenginleştirme kullanarak Niceleme Kütle Spektrometresi ile birleştiğinde
1Clinical Research Division, Fred Hutchinson Cancer Research Center - FHCRC, 2Department of Biochemistry and Microbiology, University of Victoria, 3Broad Institute of MIT and Harvard, 4Genome BC Proteomics Centre, University of Victoria, 5Plasma Proteome Institute
Kararlı İzotop Standartlar ve Anti-Peptid Antikorlar (SISCAPA) kendi proteinler için vekilleri olarak peptidler kantitatif ölçüm sağlamak için kararlı izotop dilüsyon kütle spektrometresi (MRM-MS) ile peptitler çiftler yakınlık zenginleştirme yakalayın. Burada kısmen otomatik bir biçimde manyetik parçacıklar kullanarak protokol açıklar.
Other articles by Eric Kuhn on PubMed
Posttraumatic Stress Disorder and Psychosocial Functioning Within Two Samples of MVA Survivors
To examine criterion F variables of PTSD, the psychosocial functioning of two samples of motor vehicle accident (MVA) survivors was investigated. Within each sample, comparisons between MVA survivors with and without PTSD were conducted on four psychosocial functioning indices at three time points. In addition, the relationships between specific PTSD symptom clusters and psychosocial functioning indices were examined. The study revealed that, in general, MVA survivors with PTSD evidenced poorer psychosocial functioning than did survivors without PTSD. The emotional numbing symptoms of PTSD emerged as the most consistent predictors of the psychosocial functioning indices. The implications of these findings to the comprehensive treatment of PTSD are discussed.
Of "crashes" and "accidents," a Comment on Stewart and Lord
A. E. Stewart and J. H. Lord (2002) call for abandoning the term motor vehicle accident and substituting motor vehicle crash on definitional and patient care grounds. We disagree on definitional grounds and because of the absence of empirical data from accident survivors to support their contentions.
Studies of the Vicarious Traumatization of College Students by the September 11th Attacks: Effects of Proximity, Exposure and Connectedness
From mid-October 2001 through the end of November 2001, we collected fairly large sets of questionnaires from undergraduates at three public universities (Albany, NY, n = 507, Augusta, GA, n = 336, Fargo, ND, n = 526 ) to assess rate of acute stress disorder (ASD) and level of ASD symptoms following the September 11th attacks, rate of current posttraumatic stress disorder (PTSD) and level of PTSD symptoms, and current level of depressive symptoms resulting from the September 11th attacks. We also gathered information on exposure to media coverage of the attacks, connectedness to the World Trade Center (WTC) and personnel there, and degree of engagement in reparative acts such as giving blood, attending vigils. We found higher levels of ASD, ASD symptoms, PTSD and PTSD symptoms as a function of geographical proximity to New York City (and within the Albany site, proximity of students' homes) and gender. Exposure (hours of TV watched) was a predictor in some instances as was connectedness to WTC victims. ASD symptoms were the strongest predictor of subsequent PTSD symptoms. Path models accounted for over 60% of the variance in PTSD symptoms.
Two Studies of Psychiatric Morbidity Among Motor Vehicle Accident Survivors 1 Year After the Crash
We assessed the psychiatric co-morbidity associated with chronic posttraumatic stress disorder (PTSD) (1-2 years) secondary to personal injury motor vehicle accidents (MVAs) in two studies. In Study 1, we compared the results of SCID assessments for 75 treatment-seeking MVA survivors (51 with PTSD and 24 with symptoms but no PTSD). In Study 2, we compared similar results among 132 MVA survivors who had been followed prospectively for 12+ months after their accidents (19 with PTSD, 32 who had PTSD but who had remitted, and 81 who never met criteria for PTSD). We found comparable levels of current co-morbid major depression (53%), any mood disorder (62-68%), generalized anxiety disorder (26%) and any anxiety disorder (42%) for both groups of participants with chronic PTSD. These rates of co-morbidity were higher than those found in non-PTSD comparison groups with similar MVA histories.
Quantification of C-reactive Protein in the Serum of Patients with Rheumatoid Arthritis Using Multiple Reaction Monitoring Mass Spectrometry and 13C-labeled Peptide Standards
A general method for the quantification of proteins in human serum was developed using mass spectrometry (MS) and stable isotope-labeled synthetic peptides as internal standards. Using this approach, C-reactive protein (CRP), a diagnostic marker of rheumatoid arthritis (RA), was detected in serum samples taken from patients with either erosive or nonerosive RA and compared to healthy individuals. Small volumes of serum samples were enriched for low-abundance proteins through the selective removal of human serum albumin (HSA), immunoglobulin G (IgG), and haptoglobin. After depletion of abundant proteins, the complexity of the protein mixture was further simplified using size exclusion chromatography (SEC) to fractionate denatured proteins into discrete molecular weight ranges. Fractions of interest containing CRP, M(r) = 25 000, were pooled, digested with trypsin, and then fixed quantities of the synthetic peptides were added to the mixture. The mixture of tryptic peptides was subsequently analyzed by nanoflow chromatography-tandem MS (nanoLC-MS/MS) using multiple-reaction monitoring (MRM) on a triple quadrupole mass spectrometer (TQ-MS). The ratio of transition ions derived from the endogenous and isotope-labeled peptides provided a quantitative measure of CRP in the original samples as assessed by independent measurement of CRP in the same patient samples using an immunoassay. The use of isotope-labeled synthetic peptides and MRM is a powerful analytical method for the prescreening of candidate protein biomarkers in human serum prior to antibody and immunoassay development.
One- and Two-year Prospective Follow-up of Cognitive Behavior Therapy or Supportive Psychotherapy
We followed up over 90% of 57 motor vehicle accident survivors, who completed a controlled comparison of cognitive behavioral therapy (CBT) to supportive psychotherapy (SUPPORT). One-year results showed a continued significant advantage on categorical diagnosis (PTSD or not) and structured interview measures (CAPS) for CBT over SUPPORT. Other measures generally showed the same results. At two years, we were able to follow-up only 75% of one-year completers. Although there continued to be arithmetic differences favoring CBT over SUPPORT, with these attenuated samples only differences on PTSD Checklist and Impact of Event Scale scores and in overall categorical diagnoses were significant. There was very modest improvement from end of treatment to the two-year follow-up.
Use of Mass Spectrometry to Identify Protein Biomarkers of Disease Severity in the Synovial Fluid and Serum of Patients with Rheumatoid Arthritis
To identify a panel of candidate protein biomarkers of rheumatoid arthritis (RA) that can predict which patients will develop erosive, disabling disease.
A Fluorescence-based Coupling Reaction for Monitoring the Activity of Recombinant Human NAD Synthetase
NAD synthetase is responsible for the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. This reaction provides a biosynthetic route of the coenzyme and, thus, a source of cellular reducing equivalents. Alterations in the oxidative reductive potential of the cell have been implicated as a contributing factor in many disease states. Thus, this enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a coupled enzyme assay format for the measurement of recombinant human NAD synthetase by employing lactate dehydrogenase in a cycling/amplification reaction linked ultimately to the fluorescence generation of resorufin from resazurin via diaphorase. We present kinetics of the reaction of NAD synthetase in the coupled assay format, optimization conditions, and inhibition of the reaction by gossypol [1,1',6,6',7,7'-hexahydroxy-3,3'-dimethyl-5,5'-bis(1-methylethyl)-[2,2'- binaphthalene]-8,8'-dicarboxaldehyde] and illustrate the robustness of the assay by demonstrating 384-well microtiter plate uniformity statistics. Collectively, our results show that the assay method is both robust and well suited for this class of enzymes involved in the NAD+ biosynthetic pathway.
Posttraumatic Stress and Depressive Symptoms in a College Population One Year After the September 11 Attacks: the Effect of Proximity
As a follow-up to our earlier report [Behav. Res. Ther., in press] on the level of posttraumatic stress symptoms (PTSS), depressive symptoms, and frequency of diagnoses of probable posttraumatic stress disorder (PTSD) among college students at three public universities (Albany, NY, Augusta, GA, and Fargo, ND) resulting from the September 11, 2001. Terrorist attacks, we surveyed comparable groups of students (total, n = 1313) from these three institutions in the weeks following the first anniversary (2002) of the attacks. We found proximity effects (Albany higher than Augusta which was higher than Fargo) for PTSS and depressive symptoms but not for frequency of diagnoses of probable PTSD. Within the Albany site data, proximity of county of residence to New York City (NYC) also showed a proximity effect on PTSS. Although depressive symptoms were significantly different in 2002 versus 2001, the arithmetic differences in PTSS or in frequency of diagnoses of probable PTSD were not significant. The September 11 attacks continued to exert a psychic toll on college students even a year later.
Heart Rate of Motor Vehicle Accident Survivors in the Emergency Department, Peritraumatic Psychological Reactions, ASD, and PTSD Severity: a 6-month Prospective Study
This small-scale study investigates the relationships between the heart rate of motor vehicle accident survivors presenting in the emergency department (ED) and acute stress disorder (ASD) and posttraumatic stress disorder (PTSD) symptom severity. It also examines the relationships between the survivor's heart rate in the ED and peritraumatic dissociation and peritraumatic distress reported 2 weeks posttrauma. Fifty motor vehicle accident (MVA) survivors were assessed 2 weeks, 1 (N = 42), 3 (N = 37), and 6 months (N = 37) post-MVA. The heart rate in the ED predicted self-reported ASD symptom severity and clinician-rated PTSD symptom severity at 6 months but not at 1 or 3 months. Survivors' heart rate in the ED was significantly correlated with peritraumatic dissociation but not peritraumatic distress. These findings support the role of elevated ED heart rate as a predictor of both ASD and chronic PTSD symptom severity and may help to clarify the discrepant findings of previous research.
A Controlled Evaluation of Group Cognitive Therapy in the Treatment of Irritable Bowel Syndrome
We randomized, at two sites, 210 patients with Rome II diagnosed irritable bowel syndrome (IBS), of at least moderate severity, to one of three conditions: group-based cognitive therapy (CT; n=120), psychoeducational support groups (n=46) as an active control, or intensive symptom and daily stress monitoring (n=44). One hundred eighty-eight participants completed the initial treatment. Those in symptom monitoring were then crossed over to CT. For an intent to treat analysis on a composite GI symptom measure derived from daily symptom diaries, both CT and the psychoeducational support groups were significantly more improved than those in the intensive symptom monitoring condition, but the CT and psychoeducational support group did not differ. Among treatment completers on the same composite measure of GI symptoms, again, both CT and psychoeducational support groups were statistically superior to symptom monitoring but did not differ on the symptom composite, or on any other measure. On individual IBS symptoms, both CT and psychoeducational support were statistically superior to symptom monitoring on reductions in abdominal pain and tenderness and for flatulence. Patient global ratings at the end of treatment showed the two active conditions statistically superior to symptom monitoring on change in Bowel Regularity, with CT superior to symptom monitoring on reduction in overall pain and in improvement in sense of well-being. Three-month follow-up data on 175 patients revealed maintenance of significant improvement or continued significant improvement on all IBS symptoms, including the McGill Pain Questionnaire. Group CT and psychoeducational support groups continued not to differ on any measure. We thus conclude that group CT is not superior to an attention placebo control condition.
Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution
Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1-10 ng/ml range with percent coefficients of variation from 3 to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quantitative multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a limit of quantitation in the 1-10 ng/ml range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range. These levels of assay performance represent up to a 1000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.
The Role of Stress in Symptom Exacerbation Among IBS Patients
Over 200 treatment-seeking irritable bowel syndrome (IBS) patients completed 4 weeks of daily prospective measures of stress and gastrointestinal symptoms as well as retrospective measures of stress (life events over 12 months, hassles over 1 month). We also obtained the stress measures on 66 nonill controls. Irritable bowel syndrome patients report more frequent hassles than controls and a greater stress impact than controls. Using structural equation modeling, we found that the data were consistent with a model of robust autocorrelation effects of both week-to-week gastrointestinal (GI) symptom indices (r=.84) and stress indices (r=.73), as well as strong concurrent effects of stress on IBS symptoms (r=.90) and vice versa (r=.41). The data also were consistent with a model where there were effects of stress in Week t upon GI symptoms in Week t+1 and t+2, but they were mediated through the concurrent week effects and/or autocorrelation effects. There were no statistically significant independent pathways from stress in Week t to GI symptoms in Week t+1 or t+2. Thus, there is more support for a reciprocal relation between stress and symptoms than there is for a causal relation.
Substance Abuse-related Mortality Among Middle-aged Male VA Psychiatric Patients
This study evaluated mortality and causes of death over a seven-year period among middle-aged male psychiatric patients with and without co-occurring substance use disorder.
Developing Multiplexed Assays for Troponin I and Interleukin-33 in Plasma by Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry
Protein biomarker candidates from discovery proteomics must be quantitatively verified in patient samples before they can progress to clinical validation. Here we demonstrate that peptide immunoaffinity enrichment coupled with stable isotope dilution mass spectrometry (SISCAPA-MRM) can be used to configure assays with performance suitable for candidate biomarker verification. As proof of principle, we configured SISCAPA assays for troponin I (cTnI), an established biomarker of cardiac injury, and interleukin 33 (IL-33), an emerging immunological and cardiovascular marker for which robust immunoassays are currently not available.
Quantification of Cardiovascular Biomarkers in Patient Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution
Verification of candidate biomarkers requires specific assays to selectively detect and quantify target proteins in accessible biofluids. The primary objective of verification is to screen potential biomarkers to ensure that only the highest quality candidates from the discovery phase are taken forward into preclinical validation. Because antibody reagents for a clinical grade immunoassay often exist for a small number of candidates, alternative methodologies are required to credential new and unproven candidates in a statistically viable number of serum or plasma samples. Using multiple reaction monitoring coupled with stable isotope dilution MS, we developed quantitative, multiplexed assays in plasma for six proteins of clinical relevance to cardiac injury. The process described does not require antibodies for immunoaffinity enrichment of either proteins or peptides. Limits of detection and quantitation for each signature peptide used as surrogates for the target proteins were determined by the method of standard addition using synthetic peptides and plasma from a healthy donor. Limits of quantitation ranged from 2 to 15 ng/ml for most of the target proteins. Quantitative measurements were obtained for one to two signature peptides derived from each target protein, including low abundance protein markers of cardiac injury in the nanogram/milliliter range such as the cardiac troponins. Intra- and interassay coefficients of variation were predominantly <10 and 25%, respectively. The configured multiplex assay was then used to measure levels of these proteins across three time points in six patients undergoing alcohol septal ablation for hypertrophic obstructive cardiomyopathy. These results are the first demonstration of a multiplexed, MS-based assay for detection and quantification of changes in concentration of proteins associated with cardiac injury in the low nanogram/milliliter range. Our results also demonstrate that these assays retain the necessary precision, reproducibility, and sensitivity to be applied to novel and uncharacterized candidate biomarkers for verification of proteins in blood.
Aggressive and Unsafe Driving in Male Veterans Receiving Residential Treatment for PTSD
Aggressive and unsafe driving was examined in 474 male veterans receiving Veterans Affairs residential treatment for posttraumatic stress disorder (PTSD). Specifically, the authors evaluated if PTSD was associated with aggressive and unsafe driving and if Iraq and Afghanistan War veterans were at higher risk than other war veterans. Approximately two thirds of the sample reported lifetime aggressive driving and one third reported current aggressive driving. Posttraumatic stress disorder severity was associated with aggressive driving, but not other forms of unsafe driving. Iraq and Afghanistan veterans endorsed higher rates of and more frequent aggressive driving than did other veterans. After accounting for PTSD severity, age, income, and marital status being an Iraq and Afghanistan War veteran predicted aggressive driving frequency and infrequent seatbelt use.
Evaluation of Large Scale Quantitative Proteomic Assay Development Using Peptide Affinity-based Mass Spectrometry
Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/μl in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays.
Inter-laboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma
The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to MRM-MS produces immuno-MRM assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the inter-laboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay CV, LOD, LOQ and recovery was evaluated. Limits of detection were at or below 1 ng/mL for the assayed proteins in 30 uL of plasma. Assay reproducibility was acceptable for verification studies, with median intra- and inter-laboratory CVs above the LOQ of 11% and <14%, respectively, for the entire immuno-MRM-MS assay process, including enzymatic digestion of plasma. Trypsin digestion and its requisite sample handling contributed the most to assay variability and reduced the recovery of target peptides from digested proteins. Using a stable isotope labeled protein as an internal standard instead of stable isotope labeled peptides to account for losses in the digestion process nearly doubled assay accuracy for this while improving assay precision 5%. Our results demonstrate that multiplexed immuno-MRM-MS can be made reproducible across independent laboratories and has the potential to be adopted widely for assaying proteins in matrices as complex as plasma.
