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In JoVE (1)
- मास स्पेक्ट्रोमेट्री के साथ युग्मित पेप्टाइड Immunoaffinity संवर्धन का उपयोग प्रोटीन की मात्रा का ठहराव
Other Publications (31)
- Nursing Standard (Royal College of Nursing (Great Britain) : 1987)
- Health Expectations : an International Journal of Public Participation in Health Care and Health Policy
- The American Journal of Gastroenterology
- The Journal of Biological Chemistry
- The Journal of Biological Chemistry
- Emergency Medicine Australasia : EMA
- The Journal of Primary Prevention
- AIDS Research and Human Retroviruses
- Nursing Standard (Royal College of Nursing (Great Britain) : 1987)
- Nursing Standard (Royal College of Nursing (Great Britain) : 1987)
- Current HIV Research
- Community Practitioner : the Journal of the Community Practitioners' & Health Visitors' Association
- Nurse Researcher
- PloS One
- Journal of the American Geriatrics Society
- Journal of General Internal Medicine
- PLoS Neglected Tropical Diseases
- Molecular & Cellular Proteomics : MCP
- Molecular & Cellular Proteomics : MCP
- Heart & Lung : the Journal of Critical Care
- Nature Biotechnology
- BMC Medical Education
- Health Promotion International
- Journal of Proteome Research
- Journal of Immunological Methods
- Revista De Enfermería (Barcelona, Spain)
- Cancer Research
- Clinics in Laboratory Medicine
- Molecular & Cellular Proteomics : MCP
- Primary Health Care Research & Development
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Articles by Angela Jackson in JoVE
मास स्पेक्ट्रोमेट्री के साथ युग्मित पेप्टाइड Immunoaffinity संवर्धन का उपयोग प्रोटीन की मात्रा का ठहराव
Lei Zhao*1, Jeffrey R. Whiteaker*1, Matthew E. Pope2, Eric Kuhn3, Angela Jackson4, N. Leigh Anderson5, Terry W. Pearson2, Steven A. Carr3, Amanda G. Paulovich1
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
स्थिर आइसोटोप मानक और विरोधी पेप्टाइड एंटीबॉडी (SISCAPA) जोड़े स्थिर आइसोटोप कमजोर पड़ने मास स्पेक्ट्रोमेट्री (MRM एमएस) के साथ पेप्टाइड्स उनके संबंधित प्रोटीन के लिए surrogates के रूप में पेप्टाइड्स के मात्रात्मक माप प्रदान की आत्मीयता संवर्धन द्वारा कैप्चर. यहाँ हम एक आंशिक रूप से स्वचालित प्रारूप में चुंबकीय कणों का उपयोग प्रोटोकॉल का वर्णन.
Other articles by Angela Jackson on PubMed
Nursing Standard (Royal College of Nursing (Great Britain) : 1987). Nov 26-Dec 2, 2003 | Pubmed ID: 14689664
BACKGROUND: The potential risks for and implications of black community practitioners working in racist areas is the subject of this article. It provides an overview of the risk of racial harassment and discusses the legal and ethical implications for NHS employees. This article has been written as a result of the author being confronted with the possibility of being sent to work in an area known to be racist. The British National Party's (BNP) recent gains in local elections demonstrate that such areas exist in the UK's towns and cities (BBC News 2003). CONCLUSION: The author argues that employers should do all in their power to benefit their employees and this means protecting them from working in racist areas or with racist clients who pose a potential risk.
Health Expectations : an International Journal of Public Participation in Health Care and Health Policy. Dec, 2003 | Pubmed ID: 15040796
The project aims were to enable young people to contribute their views about health services, to encourage professionals and policy makers to listen to the young people and to stimulate action to address the issues raised.
A Novel Educational Strategy to Enhance Internal Medicine Residents' Familial Colorectal Cancer Knowledge and Risk Assessment Skills
The American Journal of Gastroenterology. Mar, 2005 | Pubmed ID: 15743368
Internal medicine residents are deficient in their knowledge about familial colorectal cancer (CRC) and thus unable to comply with appropriate screening guidelines. The objective of this study was to evaluate the effectiveness of a mixed educational program that incorporates both a didactic lecture (DL) and interactive, case-based seminar (ICBS), plus distribution of a personal digital assistant (PDA)-based risk assessment tool.
The Journal of Biological Chemistry. Oct, 2005 | Pubmed ID: 16131488
Transcription of human immunodeficiency virus (HIV)-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of the C-terminal domain of RNA polymerase-II by CDK9/cyclin T1. We previously showed that Tat-induced HIV-1 transcription is regulated by protein phosphatase-1 (PP1). In the present study we demonstrate that Tat interacts with PP1 and that disruption of this interaction prevents induction of HIV-1 transcription. We show that PP1 interacts with Tat in part through the binding of Val36 and Phe38 of Tat to PP1 and that Tat is involved in the nuclear and subnuclear targeting of PP1. The PP1 binding mutant Tat-V36A/F38A displayed a decreased affinity for PP1 and was a poor activator of HIV-1 transcription. Surprisingly, Tat-Q35R mutant that had a higher affinity for PP1 was also a poor activator of HIV-1 transcription, because strong PP1 binding competed out binding of Tat to CDK9/cyclin T1. Our results suggest that Tat might function as a nuclear regulator of PP1 and that interaction of Tat with PP1 is critical for activation of HIV-1 transcription by Tat.
The Journal of Biological Chemistry. Jan, 2006 | Pubmed ID: 16291746
Native uncoupling protein 1 (UCP 1) was purified from rat mitochondria by hydroxyapatite chromatography and identified by peptide mass mapping and tandem mass spectrometry. Native and expressed UCP 1 were reconstituted into liposomes, and proton flux through UCP 1 was shown to be fatty acid-dependent and GDP-sensitive. To investigate the mechanism of action of UCP 1, we determined whether hydrophilic modification of the omega-carbon of palmitate effected its transport function. We show that proton flux was greater through native UCP 1-containing proteoliposomes when facilitated by less hydrophilically modified palmitate (palmitate > omega-methoxypalmitate > omega-hydroxypalmitate with little or no proton flux due to glucose-O-omega-palmitate or undecanesulfonate). We show that non-proton-dependent charge transfer was greater when facilitated by less hydrophilically modified palmitate (palmitate/undecanesulfonate > omega-methoxypalmitate > omega-hydroxypalmitate, with no non-proton-dependent charge transfer flux due to glucose-O-omega-palmitate). We show that the GDP-inhibitable oxygen consumption rate in brown adipose tissue mitochondria was reversed by palmitate (as expected) but not by glucose-O-omega-palmitate. Our data are consistent with the model that UCP 1 flips long-chain fatty acid anions and contradict the "cofactor" model of UCP 1 function.
Emergency Medicine Australasia : EMA. Apr, 2006 | Pubmed ID: 16669947
Within a few short months of the tsunami, an earthquake measuring 8.7 on the Richter scale devastated the Indonesian island of Nias. The present paper describes the experiences of two emergency medicine staff deployed to the island operating in small teams on the remote western part of the island. It discusses the benefits of utilizing experienced emergency medicine staff and looks at lessons learnt from the deployment.
Uncovering the "skeleton in the Closet": the Issue of Bone and Joint Disorders in the Maldives and the Opportunities for Primary Prevention and Health Promotion
The Journal of Primary Prevention. Jul, 2006 | Pubmed ID: 16767340
Disorders of bones and joints are the most common cause of severe long-term pain and disability around the world yet receive inadequate attention in many countries. Bone/joint health is absent from current health policy and the primary prevention/health promotion agenda in Maldives and diagnostic and curative infrastructure is very limited. In 2004, tentative evidence emerged indicating that degenerative bone and joint conditions may impose a large burden on community health. A study was undertaken amongst a representative community sample of women aged 15-50 to further investigate the issue. One third reported bone/joint problems; very high proportions manifested a range of osteoporosis risk factors, and sizeable numbers appear to be at elevated risk of osteoarthritis. Bone/joint health knowledge was very limited. The findings suggest strong potential for primary prevention on modifiable risk factors, need for research with other population groups, and development of screening and curative care. EDITORS' STRATEGIC IMPLICATIONS: The author provides important data on the burden of disorders of bones/joints on a "developing" community and presents recommendations for behavioral and educational prevention strategies that may prove useful across societies.
New CD4+ and CD8+ T Cell Responses Induced in Chronically HIV Type-1-infected Patients After Immunizations with an HIV Type 1 Lipopeptide Vaccine
AIDS Research and Human Retroviruses. Jul, 2006 | Pubmed ID: 16831093
We showed that an anti-HIV lipopeptide vaccine injected to HIV-uninfected volunteers was well tolerated and able to induce a specific CD4(+) and CD8(+) T cell responses. The same vaccine was injected in HIV-1 chronically infected patients controlled by HAART to evaluate its immunogenicity. In this trial, 24 patients were immunized three times with a mixture of six lipopeptides (Nef 66-97, Nef 117-147, Nef 182-205, Gag 183-214, Gag 253-284, and Env 303-335) at 0, 3, and 6 weeks. We studied the HIV-1-specific CD4(+) T cell proliferative responses. The IFN-gamma secretion by activated CD8(+) T cells was evaluated, using an ex vivo ELISpot assay and 60 CD8(+) T cell epitopes derived from the vaccine. Before immunization (W0), anti-HIV CD4(+) T cell responses to Gag, Nef, and Env large peptides were detected in 7/23 (30%) analyzable patients. After three injections, 17/23 (74%) patients had a proliferative response and 16 of them induced new specific CD4(+) T cell responses. At W0, CD8(+) T cell responses to HIV-1 epitopes were detected in 6/23 (26%) patients. After vaccination, 16/23 (70%) patients showed CD8(+) T cell responses and 13 of these patients induced new T cell responses to 25 different HIV-1 epitopes. These HIV-1 epitopes were detected in patients with various HLA class I molecules (HLA-A2, -A3/A11, -A24, -B7 superfamily, -B8), as found in the majority of the white population. Lipopeptides induce new anti-HIV T cell responses in vaccinated infected patients and could be used as a new immunotherapy strategy. The majority of these responders induced specific new CD4(+) and CD8(+) T cell responses.
Nursing Standard (Royal College of Nursing (Great Britain) : 1987). Jul 5-11, 2006 | Pubmed ID: 16875280
Nursing Standard (Royal College of Nursing (Great Britain) : 1987). Aug 30-Sep 5, 2006 | Pubmed ID: 16972576
Retrovirology. 2006 | Pubmed ID: 17083724
Transcription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription.
Current HIV Research. Jan, 2007 | Pubmed ID: 17266553
The emergence of drug-resistant HIV-1 strains presents a challenge for the design of new drugs. Targeting host cell factors involved in the regulation of HIV-1 replication might be one way to overcome the resistance of HIV-1 to anti-viral agents. Our recent studies identified protein phosphatase-1 (PP1) as an important regulator of HIV-1 transcription. Transcription of HIV-1 genes is activated by HIV-1 Tat protein that induces phosphorylation of the C-terminal domain of RNA polymerase-II by CDK9/cyclin T1. We have shown that HIV-1 Tat binds PP1 in vitro; targets PP1 to the nucleus; and that Tat interaction with PP1 is important for HIV-1 transcription. In this review, we discuss two potential targets of PP1 in Tat-induced HIV-1 transcription: the C-terminal domain of RNA polymerase-II and CDK9. We also present a computer model of Tat-PP1 complex that might be useful for future drug design in anti-HIV-1 therapeutics.
Community Practitioner : the Journal of the Community Practitioners' & Health Visitors' Association. Feb, 2007 | Pubmed ID: 17330669
The aim of this research study is to explore health visitors' beliefs, knowledge and practice in cultural competence. A baseline survey was undertaken with all health visitors working within a West Midlands primary care trust (PCT) which served a significant population of minority ethnic communities. The results show that half the respondents were themselves members of a minority ethnic community. Caribbean origins predominated, with little representation from those of Asian descent. Non-parametric testing indicated that respondents showed there was a significant difference in their ability to meet the needs of minority ethnic communites as opposed to their ability to meet the white population needs. Respondents were able to identify factors which promote, and barriers which hamper use of health visiting services by minority ethnic communities, for example, the standard yet important factors of language and culture. However, racism was not recognised as a significant issue. The need for cultural competence training was seen as a key outcome.This training must reflect the diverse cultural needs of staff and service users.
Nurse Researcher. 2007 | Pubmed ID: 17702146
Angela Knight Jackson, acting R&D lead nurse, Heart of Birmingham Teaching Primary Care Trust, talks about the factors that have contributed to her success.
Cellular Immune Responses Induced with Dose-sparing Intradermal Administration of HIV Vaccine to HIV-uninfected Volunteers in the ANRS VAC16 Trial
PloS One. 2007 | Pubmed ID: 17712402
The objective was to compare the safety and cellular immunogenicity of intradermal versus intramuscular immunization with an HIV-lipopeptide candidate vaccine (LIPO-4) in healthy volunteers.
Chief Resident Immersion Training in the Care of Older Adults: an Innovative Interspecialty Education and Leadership Intervention
Journal of the American Geriatrics Society. Jun, 2008 | Pubmed ID: 18410320
Chief residents (CRs) play a crucial role in training residents and students but may have limited geriatrics training or formal preparation for their CR role. A 2-day off-site chief resident immersion training (CRIT) addressed these challenges. Objectives were to foster collaboration between disciplines in the management of complex older patients, increase knowledge of geriatrics principles to incorporate into teaching, enhance leadership skills, and help CRs develop an achievable project for implementation in their CR year. Three cohorts totaling 47 trainees and 18 faculty mentors from 13 medical and surgical disciplines participated over 3 successive years. The curriculum, developed and taught by a multidisciplinary team, featured an interactive surgical case, mini-lectures on geriatrics topics, seminars to enhance teaching and leadership skills, and one-on-one mentoring to develop a project in geriatric care or education. Evaluation included pre- and postprogram tests and self-report surveys and two follow-up surveys or interviews. In 2006 and 2007, scores on a 12-item objective knowledge test increased significantly (P<.001) from before to immediately after CRIT. Self-report knowledge and confidence in teaching geriatrics also increased significantly (P<.05) in all formally covered topics. Mean enhancement of CR skills was 4.3 (1=not at all, 5=very much). Eleven months after CRIT, all but five CRs had implemented at least part of their action projects. CRs reported improved care of older patients, better leadership skills, more and better geriatrics teaching, and more collaboration between disciplines. A 2-day interactive program for CRs can increase institutional capacity regarding geriatrics teaching and care of elderly patients across medical specialties.
Promoting Substance Use Education Among Generalist Physicians: an Evaluation of the Chief Resident Immersion Training (CRIT) Program
Journal of General Internal Medicine. Jan, 2009 | Pubmed ID: 18937015
Education about substance use (SU) disorders remains inadequate in medical training.
PLoS Neglected Tropical Diseases. 2009 | Pubmed ID: 19190729
Tropical diseases caused by parasites continue to cause socioeconomic devastation that reverberates worldwide. There is a growing need for new control measures for many of these diseases due to increasing drug resistance exhibited by the parasites and problems with drug toxicity. One new approach is to apply host defense peptides (HDP; formerly called antimicrobial peptides) to disease control, either to treat infected hosts, or to prevent disease transmission by interfering with parasites in their insect vectors. A potent anti-parasite effector is bovine myeloid antimicrobial peptide-27 (BMAP-27), a member of the cathelicidin family. Although BMAP-27 is a potent inhibitor of microbial growth, at higher concentrations it also exhibits cytotoxicity to mammalian cells. We tested the anti-parasite activity of BMAP-18, a truncated peptide that lacks the hydrophobic C-terminal sequence of the BMAP-27 parent molecule, an alteration that confers reduced toxicity to mammalian cells.
Molecular & Cellular Proteomics : MCP. May, 2009 | Pubmed ID: 19196707
A SISCAPA (stable isotope standards and capture by anti-peptide antibodies) method for specific antibody-based capture of individual tryptic peptides from a digest of whole human plasma was developed using a simplified magnetic bead protocol and a novel rotary magnetic bead trap device. Following off-line equilibrium binding of peptides by antibodies and subsequent capture of the antibodies on magnetic beads, the bead trap permitted washing of the beads and elution of bound peptides inside a 150-microm-inner diameter capillary that forms part of a nanoflow LC-MS/MS system. The bead trap sweeps beads against the direction of liquid flow using a continuous succession of moving high magnetic field-gradient trap regions while mixing the beads with the flowing liquid. This approach prevents loss of low abundance captured peptides and allows automated processing of a series of SISCAPA reactions. Selected tryptic peptides of alpha(1)-antichymotrypsin and lipopolysaccharide-binding protein were enriched relative to a high abundance serum albumin peptide by 1,800 and 18,000-fold, respectively, as measured by multiple reaction monitoring. A large majority of the peptides that are bound nonspecifically in SISCAPA reactions were shown to bind to components other than the antibody (e.g. the magnetic beads), suggesting that substantial improvement in enrichment could be achieved by development of improved inert bead surfaces.
Multiple Reaction Monitoring-based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma
Molecular & Cellular Proteomics : MCP. Aug, 2009 | Pubmed ID: 19411661
Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Stable isotope-labeled standard peptides were added immediately following tryptic digestion because addition of stable isotope-labeled standard peptides prior to trypsin digestion was found to generate elevated and unpredictable results. Proteotypic tryptic peptides containing isotopically coded amino acids ([(13)C(6)]Arg or [(13)C(6)]Lys) were synthesized for all 45 proteins. Peptide purity was assessed by capillary zone electrophoresis, and the peptide quantity was determined by amino acid analysis. For maximum sensitivity and specificity, instrumental parameters were empirically determined to generate the most abundant precursor ions and y ion fragments. Concentrations of individual peptide standards in the mixture were optimized to approximate endogenous concentrations of analytes and to ensure the maximum linear dynamic range of the MRM assays. Excellent linear responses (r > 0.99) were obtained for 43 of the 45 proteins with attomole level limits of quantitation (<20% coefficient of variation) for 27 of the 45 proteins. Analytical precision for 44 of the 45 assays varied by <10%. LC-MRM/MS analyses performed on 3 different days on different batches of plasma trypsin digests resulted in coefficients of variation of <20% for 42 of the 45 assays. Concentrations for 39 of the 45 proteins are within a factor of 2 of reported literature values. This mixture of internal standards has many uses and can be applied to the characterization of trypsin digestion kinetics and plasma protein expression profiling because 31 of the 45 proteins are putative biomarkers of cardiovascular disease.
Self-help Groups for Patients with Coronary Heart Disease As a Resource for Rehabilitation and Secondary Prevention-what is the Evidence?
Heart & Lung : the Journal of Critical Care. May-Jun, 2009 | Pubmed ID: 19486787
Cardiovascular heart disease (CHD) is a major health care concern worldwide. Maintaining regular cardiac rehabilitation attendance and secondary-prevention strategies are significant health care challenges. Although self-help groups provide benefit for many chronic health conditions, it is not clear if they address the challenges of CHD rehabilitation and self-management. This literature review was guided by the following question: Can self-help groups address the challenges of CHD rehabilitation and self-management? This article reviews the traditional published and "grey" literature on CHD-related self-help groups identified from a database search (Cochrane Library, PubMed, PsychINFO, Medline, Cumulative Index to Nursing and Allied Health Literature, Applied Social Sciences Index and Abstracts, and Social Sciences Citation Index). Identified articles were screened based on the type of initiative: Community-based non-health service-organized groups were included, but hospital-based group treatment and therapy interventions or programs were excluded. Published research and analysis of CHD-related self-help groups is scarce. Sixteen articles focusing on self-help groups were identified. The review results indicate that the limited quantity, limited range, and variable quality of studies prevents reliable conclusions being made regarding effects and outcomes as well as the extent and profile of participation. Strengthening the evidence base regarding the impact of CHD-related self-help groups, the reasons for participation versus nonparticipation in such groups, and determining nonparticipants support needs must be done to establish if and for which patients such groups constitute an effective resource for rehabilitation and secondary prevention.
Multi-site Assessment of the Precision and Reproducibility of Multiple Reaction Monitoring-based Measurements of Proteins in Plasma
Nature Biotechnology. Jul, 2009 | Pubmed ID: 19561596
Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low mug/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.
Internal Medicine Residency Training for Unhealthy Alcohol and Other Drug Use: Recommendations for Curriculum Design
BMC Medical Education. 2010 | Pubmed ID: 20230607
Unhealthy substance use is the spectrum from use that risks harm, to use associated with problems, to the diagnosable conditions of substance abuse and dependence, often referred to as substance abuse disorders. Despite the prevalence and impact of unhealthy substance use, medical education in this area remains lacking, not providing physicians with the necessary expertise to effectively address one of the most common and costly health conditions. Medical educators have begun to address the need for physician training in unhealthy substance use, and formal curricula have been developed and evaluated, though broad integration into busy residency curricula remains a challenge.
How Can Health Promotion Interventions Be Adapted for Minority Ethnic Communities? Five Principles for Guiding the Development of Behavioural Interventions
Health Promotion International. Jun, 2010 | Pubmed ID: 20299500
The term 'culturally sensitive' is often used to describe interventions adapted for minority ethnic communities. However, understanding of strategies for adapting behavioural interventions for such communities is limited. The questions addressed in this paper are: What are the main strategies for adapting interventions to reduce coronary heart disease (CHD) for minority ethnic communities? Why have interventions been adapted in these ways? A systematic review was carried out to investigate interventions for preventing CHD, including promoting physical activity, smoking cessation and healthier diets in Pakistani, Chinese and Indian communities in countries where these groups are minorities. International databases and key websites were searched, and 23 477 titles and abstracts were initially identified. Seventeen papers met inclusion and quality criteria. A 'meta-ethnographic' approach to data synthesis was employed to identify underlying principles for adapting interventions. The rationale underpinning adaptations is not made explicit in individual studies, limiting generalizability. Five principles for adapting behavioural interventions for minority ethnic communities were identified: (i) use community resources to publicize the intervention and increase accessibility; (ii) identify and address barriers to access and participation; (iii) develop communication strategies which are sensitive to language use and information requirements; (iv) work with cultural or religious values that either promote or hinder behavioural change; and (v) accommodate varying degrees of cultural identification. While the principles require further testing and verification, they have been generated through a systematic approach to study identification, quality appraisal and data synthesis. This represents significant progress in advancing understanding of adapted behavioural interventions for minority ethnic communities.
A Quantitative Study of the Effects of Chaotropic Agents, Surfactants, and Solvents on the Digestion Efficiency of Human Plasma Proteins by Trypsin
Journal of Proteome Research. Oct, 2010 | Pubmed ID: 20722421
Plasma biomarkers studies are based on the differential expression of proteins between different treatment groups or between diseased and control populations. Most mass spectrometry-based methods of protein quantitation, however, are based on the detection and quantitation of peptides, not intact proteins. For peptide-based protein quantitation to be accurate, the digestion protocols used in proteomic analyses must be both efficient and reproducible. There have been very few studies, however, where plasma denaturation/digestion protocols have been compared using absolute quantitation methods. In this paper, 14 combinations of heat, solvent [acetonitrile, methanol, trifluoroethanol], chaotropic agents [guanidine hydrochloride, urea], and surfactants [sodium dodecyl sulfate (SDS) and sodium deoxycholate (DOC)] were compared with respect to their effectiveness in improving subsequent tryptic digestion. These digestion protocols were evaluated by quantitating the production of proteotypic tryptic peptides from 45 moderate- to high-abundance plasma proteins, using tandem mass spectrometry in multiple reaction monitoring mode, with a mixture of stable-isotope labeled analogues of these proteotypic peptides as internal standards. When the digestion efficiencies of these 14 methods were compared, we found that both of the surfactants (SDS and DOC) produced an increase in the overall yield of tryptic peptides from these 45 proteins, when compared to the more commonly used urea protocol. SDS, however, can be a serious interference for subsequent mass spectrometry. DOC, on the other hand, can be easily removed from the samples by acid precipitation. Examining the results of a reproducibility study, done with 5 replicate digestions, DOC and SDS with a 9 h digestion time produced the highest average digestion efficiencies (∼80%), with the highest average reproducibility (<5% error, defined as the relative deviation from the mean value). However, because of potential interferences resulting from the use of SDS, we recommend DOC with a 9 h digestion procedure as the optimum protocol.
MALDI Immunoscreening (MiSCREEN): a Method for Selection of Anti-peptide Monoclonal Antibodies for Use in Immunoproteomics
Journal of Immunological Methods. Feb, 2011 | Pubmed ID: 21078325
A scalable method for screening and selection of peptide-specific monoclonal antibodies (mAbs) is described. To identify high affinity anti-peptide mAbs in hybridoma supernatants, antibodies were captured by magnetic affinity beads followed by binding of specific peptides from solution. After timed washing steps, the remaining bound peptides were eluted from the beads and detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). This allowed measurement of monovalent interactions of peptides with single antigen binding sites on the antibodies, thus reflecting antibody affinity rather than avidity. Antibodies that were able to bind target peptides from solution phase and retain them during washing for a minimum of 10 min were identified by the strength of the appropriate m/z peptide MS signals obtained. This wash time reflects the minimum peptide dissociation time required for use of these antibodies in several current immuno-mass spectrometry assays. Kinetic analysis of antibody-peptide binding by surface plasmon resonance (SPR) showed that the selected antibodies were of high affinity and, most importantly, had low dissociation constants. This method, called MALDI immunoscreening (MiSCREEN), thus enables rapid screening and selection of high affinity anti-peptide antibodies that are useful for a variety of immunoproteomics applications. To demonstrate their functional utility in immuno-mass spectrometry assays, we used the selected, purified RabMAbs to enrich natural (tryptic) peptides from digested human plasma.
Revista De Enfermería (Barcelona, Spain). Jan, 2011 | Pubmed ID: 21428012
Cancer Research. Sep, 2011 | Pubmed ID: 21742770
Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors.
High-flow Multiplexed MRM-based Analysis of Proteins in Human Plasma Without Depletion or Enrichment
Clinics in Laboratory Medicine. Sep, 2011 | Pubmed ID: 21907103
Inter-laboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma
Molecular & Cellular Proteomics : MCP. Dec, 2011 | Pubmed ID: 22199228
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.
A Qualitative Study Exploring Why People Do Not Participate in Cardiac Rehabilitation and Coronary Heart Disease Self-help Groups, and Their Rehabilitation Experience Without These Resources
Primary Health Care Research & Development. Jan, 2012 | Pubmed ID: 21819643
Secondary prevention and self-management of coronary heart disease (CHD) are of major importance to people who survive myocardial infarction (MI). This can be facilitated by cardiac rehabilitation (CR; the formal health service programme) and informal CHD self-help groups. Non-participation is an important issue, yet it is poorly understood. Rehabilitation difficulties and prevention challenges have been identified among people following MI, but the particular experience and perspective of CR and CHD group non-participants are largely unknown.