Promoting community engagement in spinal cord injury research: a case example.

Community engagement is an important method of knowledge translation in spinal cord injury (SCI) research where researchers collaborate with people with lived experience, care partners, and other research users to improve the quality of research. This perspective article aims to promote community engagement in SCI research by describing useful resources for its implementation and providing an example project using the North American Spinal Cord Injury Consortium (NASCIC) process for such partnerships. Researchers from the Jefferson College of Rehabilitation Sciences' (JCRS) Center for Outcomes and Measurement engaged NASCIC to create an advisory committee composed of four people living with SCI to make recommendations for the methods of a large-scale study to develop a clinical outcome assessment. The advisory committee made usable recommendations for enhancing recruitment methods and reducing burden and barriers to participation. The successful partnership between NASCIC and JCRS shows the feasibility and value of SCI community engagement in research.

An Assessment of Which Sociodemographic and Spinal Cord Injury-Specific Characteristics Influence Engagement With Experimental Therapies and Participation in Clinical Trials.

Background: Although a number of experimental therapies for spinal cord injury (SCI) have recently emerged, few authors have examined the goals of individuals with SCI considering experimental therapies, and none have determined whether sociodemographic and injury-specific characteristics influence that engagement. Objectives: To determine (a) the goals of individuals with SCI who are considering experimental therapies; (b) whether sociodemographic factors, injury-specific characteristics, and concerns over adverse events influence those goals and/or participation in experimental therapies and clinical trials; and (c) whether people with SCI feel they have adequate information about experimental therapies and clinical trials. Methods: An online survey that yielded 364 responses. Results: Most respondents (83.7%) had sought information about experimental therapies, and just under half (47.8%) had received one. The most frequently cited functional goals were improvement in bowel and bladder function and elimination of dysreflexia (60.4%). Several goals were influenced by age and level and completeness of injury, and most respondents (93.4%) wanted more information about experimental therapies. Just over one-third (34.6%) of respondents had participated in a clinical trial, and nearly all (96.9%) wanted more information about them. Having received experimental therapies and participated in clinical trials was positively correlated with seeking SCI-specific care from an SCI specialist rather than from a primary care physician. Most (83.9%) respondents would avoid or be reluctant to engage with a medical center if they were made aware of harm done to trial participants. Conclusion: This work suggests that there are unmet information needs among people with SCI, specifically pertaining to experimental therapies and clinical trials. It also reveals that improved access to SCI specialists may enhance access to novel treatments and research efforts. Being made aware of harm to trial participants may influence the decision of individuals with SCI to seek care at or enroll in trials at these clinical sites.

How individuals with spinal cord injury in the United States access and assess information about experimental therapies and clinical trials: results of a clinical survey.

STUDY DESIGN: An internet-based survey. OBJECTIVES: To determine how individuals with spinal cord injury (SCI) access information about experimental therapies and clinical trials. To understand which factors influence receipt of and perceived trustworthiness of that information. SETTING: Two academic medical centers and an SCI organization. METHODS: Demographic information frequencies and percentages were calculated then analyzed using chi-square tests for independence. Fisher's exact test of independence was used to assess significance for contingency tables with categories containing expected counts below five. RESULTS: Three hundred sixty four persons with SCI participated in the survey. Most felt confident in their ability to evaluate SCI-specific information from a variety of sources, though SCI organizations and the medical literature were deemed the most reliable. Information from SCI specialists was deemed more credible than that from non-SCI specialists, but only 53.6% of participants had access to them. Nearly all (89.0%) respondents who had sought information about experimental therapies had found it online, while 51.4% of those who had participated in a clinical trial had been contacted by a research team. Only 8.4% of participants felt their medical teams offered them sufficient information about experimental therapies and clinical trials. Wealthier and more educated respondents were more knowledgeable about health-related resources on the internet. Nearly all participants (96.9%) expressed interest in learning more about trials related to SCI. CONCLUSIONS: There is an information deficit among people with SCI pertaining to experimental therapies and clinical trials. It is exacerbated by lack of income, education, and access to SCI specialists.

Community-Based Dialysis in Saskatchewan First Nations: A Grassroots Approach to Gaining Insight and Perspective From First Nations Patients With Chronic Kidney Disease.

BACKGROUND: Renal replacement options or dialysis can be delivered in the home setting or hospital setting. Home dialysis offers a number of benefits over hospital-delivered dialysis. These advantages include improved quality of life, less travel, and fewer dietary restrictions. Despite the benefits, home-based dialysis therapies are significantly underutilized by First Nations with only 16.2% uptake versus 25.7% uptake in non-First Nations people in Saskatchewan. It is important to recognize that First Nations have a greater burden of end-stage renal disease including higher prevalence, younger age at diagnosis, increased severity of disease, mortality at an earlier age, and increased travel distance to access kidney services. OBJECTIVE: The goal of this study is to identify the existing barriers to home peritoneal dialysis and provide insight for future programs in Saskatchewan First Nations communities in a culturally meaningful framework. DESIGN: Through qualitative research utilizing sharing circles and individual interviews, barriers to utilizing home-based dialysis were identified. SETTING: Four sharing circles were held and interviews were conducted with four First Nations dialysis patients. PARTICIPANTS: Total number of participants in sharing circles were 67. Sharing circles were composed of patients with chronic kidney disease, patients on hospital-based dialysis, patients on home-based peritoneal dialysis, family members, health care providers (nurses, physicians, dietitians, primary care director, and coordinators). Face-to-face interviews were conducted with four First Nations dialysis patients. MEASUREMENTS: The data from the sharing circles and interviews were transcribed and analyzed by a PhD researcher using constructivist grounded theory, with elements of narrative inquiry to ascertain participants' experiences of care. Data were coded and then grouped into categories using qualitative research software NVivo. Saturation of data was achieved. METHODS: Documenting and recounting patient and community experience with chronic kidney disease through sharing circles involving patients, family members, and health care providers has been the central information base for this project. Qualitative interviews were conducted with patients who currently use home dialysis and those who travel to hospital for dialysis. Written consent was obtained from all participants. Information was gathered via audio recording of all sharing circles and interviews. Transcription of the interviews was completed with confidentiality maintained during transcription. RESULTS: The main theme of our results was addressing the underutilization of home-based peritoneal dialysis in First Nations Communities. Five subthemes emerged from the main theme and included logistics, education and information, training and support, community support, and culture and leadership. Through sharing circles, a secondary theme of observations about living with chronic kidney disease and experiences of being on dialysis was explored. LIMITATIONS: A small number of First Nations communities were involved in this project, and although the data reached saturation, we cannot presume that the information is representative of all First Nations in Saskatchewan. There were a limited number of patients currently on home-based peritoneal dialysis, and therefore their perceptions may not be adequately captured. Participant characteristics (patient, caregiver, nurse, etc) were not captured when speaking in the sharing circles, and therefore participants are not classified when quoted. CONCLUSIONS: Strategies to help improve home-based dialysis included improved education, local support, integrated traditional medicine, cultural sensitivity, and leadership prioritization.

CONTEXTE: La dialyse et les autres modalités de remplacement rénal sont offertes en centre ou au domicile du patient. La dialyse à domicile offre de nombreux avantages comparativement à la dialyse en centre, notamment une meilleure qualité de vie, et requiert moins de déplacements et de restrictions alimentaires. Néanmoins, cette modalité continue d'être sous-utilisée par les patients autochtones en Saskatchewan (16,2 % contre 25,7 % dans la population allochtone). Il convient de souligner que le fardeau de l'insuffisance rénale terminale est plus important chez les patients autochtones: prévalence plus élevée, diagnostic et mortalité plus précoces, sévérité accrue de la maladie et distances plus grandes à parcourir pour accéder aux services néphrologiques. OBJECTIFS: L'étude vise à recenser les facteurs limitant l'adoption de la dialyse péritonéale à domicile comme modalité, et à fournir un aperçu des programmes à venir dans les communautés des Premières Nations de la Saskatchewan dans un cadre culturellement significatif. TYPE D'ÉTUDE: Nous avons recensé les obstacles au choix de la dialyse à domicile comme modalité de traitement par l'entremise d'une recherche qualitative fondée sur des cercles de partage et des entretiens individuels. CADRE: Quatre cercles de partage ont été tenus et des entretiens ont été conduits auprès de quatre patients autochtones dialysés. PARTICIPANTS: Au total, 67 personnes ont participé aux cercles de partage composés de patients atteints d'insuffisance rénale chronique, de patients dialysés en centre, de patients dialysés à domicile, de membres de leurs familles et de fournisseurs de soins (infirmières, médecins, diététistes, coordinateurs, administrateurs de centres de soins de santé primaires). Quatre patients autochtones traités en dialyse ont été questionnés individuellement. MESURES: Pour catégoriser les expériences de soins, les données recueillies au cours des entretiens et des cercles de partage ont été transcrites et analysées par un chercheur doctorant à l'aide d'une théorie constructiviste fondée sur des éléments d'enquête narrative. Les données ont été codées et groupées en catégories grâce au logiciel d'analyse qualitative NVivo. La saturation des données a été atteinte. MÉTHODOLOGIE: La base d'information centrale de ce projet a été de documenter et de relater l'expérience des communautés autochtones et des patients atteints de néphropathie par l'entremise de cercles de partages impliquant leurs familles et des fournisseurs de soins. Les entretiens qualitatifs ont été menés auprès de patients pratiquant la dialyse à domicile et de patients recevant leurs traitements en centre hospitalier. Le consentement écrit a été obtenu de tous les participants. L'information a été recueillie à partir de l'enregistrement audio des entretiens et des cercles de partage. La transcription des entretiens a été complétée dans le respect de la confidentialité. RÉSULTATS: Le thème principal de notre recherche était d'aborder la sous-utilisation de la dialyse péritonéale à domicile comme modalité dans les communautés autochtones. Cinq sous-thèmes ont découlé du thème principal, soit: a) la logistique, b) l'éducation et l'information, c) la formation et le soutien, d) le soutien de la communauté, et e) la culture et le leadership. Un thème secondaire a été exploré par l'entremise des cercles de partages, soit la collecte d'observations concernant la vie avec l'insuffisance rénale chronique et l'expérience d'être traité en dialyse. LIMITES: Peu de communautés autochtones ont été impliquées dans ce projet et bien que les données aient atteint la saturation, nous ne pouvons présumer que l'information recueillie est représentative de tous les membres des Premières Nations de la Saskatchewan. Un faible nombre de patients était traité par dialyse à domicile; dès lors, leurs perceptions pourraient ne pas être saisies adéquatement. Les caractéristiques des participants (patients, fournisseurs de soins, infirmières, etc.) n'ont pas été colligées lors des cercles de partage et ainsi, les participants ne sont pas classés lorsque cités. CONCLUSION: Les stratégies visant à favoriser la dialyse à domicile incluaient l'amélioration de l'éducation, le soutien local, l'intégration des pratiques de la médecine traditionnelle, une approche tenant compte des différences culturelles et la priorisation de la part des intervenants.

A Retrospective Study of Chronic Kidney Disease Burden in Saskatchewan's First Nations People.

BACKGROUND: Chronic kidney disease is more prevalent among First Nations people than in non-First Nations people. Emerging research suggests that First Nations people are subject to greater disease burden than non-First Nations people. OBJECTIVE: We aimed to identify the severity of chronic kidney disease and quantify the geographical challenges of obtaining kidney care by Saskatchewan's First Nations people. DESIGN: This study is a retrospective analysis of the provincial electronic medical record clinical database from January 2012 to December 2013. SETTING: The setting involved patients followed by the Saskatchewan provincial chronic kidney care program, run out of two clinics, one in Regina, SK, and one in Saskatoon, SK. PATIENTS: The patients included 2478 individuals (379 First Nations and 2099 non-First Nations) who were older than 18 years old, resident in Saskatchewan, and followed by the provincial chronic kidney care program. First Nations individuals were identified by their Indigenous and Northern Affairs Canada (INAC) Number. MEASUREMENTS: The demographics, prevalence, cause of end-stage renal disease, severity of chronic kidney disease, use of home-based therapies, and distance traveled for care among patients are reported. METHODS: Data were extracted from the clinical database used for direct patient care (the provincial electronic medical record database for the chronic kidney care program), which is prospectively managed by the health care staff. Actual distance traveled by road for each patient was estimated by a Geographic Information System Analyst in the First Nations and Inuit Health Branch of Health Canada. RESULTS: Compared with non-First Nations, First Nations demonstrate a higher proportion of end-stage renal disease (First Nations = 33.0% vs non-First Nations = 21.4%, P < .001), earlier onset of chronic kidney disease (MFN = 56.4 years, SD = 15.1; MNFN = 70.6 years, SD = 14.7, P < .001), and higher rates of end-stage renal disease secondary to type 2 diabetes (First Nations = 66.1% vs non-First Nations = 39.0%, P < .001). First Nations people are also more likely to be on dialysis (First Nations = 69.7% vs non-First Nations = 40.2%, P < .001), use home-based therapies less frequently (First Nations = 16.2% vs non-First Nations = 25.7%; P = 003), and must travel farther for treatment (P < .001), with First Nations being more likely than non-First Nations to have to travel greater than 200 km. LIMITATIONS: Patients who are followed by their primary care provider or solely through their nephrologist's office for their chronic kidney disease would not be included in this study. Patients who self-identify as Aboriginal or Indigenous without an INAC number would not be captured in the First Nations cohort. CONCLUSIONS: In Saskatchewan, First Nations' burden of chronic kidney disease reveals higher severity, utilization of fewer home-based therapies, and longer travel distances than their non-First Nations counterparts. More research is required to identify innovative solutions within First Nations partnering communities.

CONTEXTE: La prévalence de l'insuffisance rénale chronique (IRC) est plus élevée chez les autochtones (AUT) que chez les allochtones (ALL); de nouvelles études indiquent que les Premières Nations seraient davantage affligés que les allochtones par le fardeau de la maladie. OBJECTIFS: Notre objectif était bipartite : i) mesurer la gravité de l'IRC chez les autochtones et; ii) quantifier le défi géographique posé par la distance que les Saskatchewanais autochtones ont à parcourir pour obtenir des soins de santé rénale. TYPE D'ÉTUDE: L'étude est une analyse rétrospective de la base de données provinciale des dossiers médicaux informatisés pour la période s'échelonnant de janvier 2012 à décembre 2013. CADRE: L'étude concerne les patients suivis dans deux cliniques saskatchewanaises (une à Régina et une autre à Saskatoon) participant au programme provincial de soins des maladies rénales chroniques. SUJETS: L'étude porte sur 2 478 patients adultes (379 autochtones et 2 099 allochtones) résidents de la Saskatchewan et suivis par le programme provincial de soins des maladies rénales chroniques. Les membres des Premières Nations ont été identifiés par leur numéro de Certificat de statut Indien (CSI) émis par le ministère des Affaires Autochtones et du Nord Canada (AADNC*). MESURES: Ont été colligées les données démographiques des patients, la prévalence de la maladie, les causes de l'insuffisance rénale terminale (IRT), la gravité de l'atteinte, le recours ou non à des traitements à domicile, et la distance à parcourir pour obtenir des soins. MÉTHODOLOGIE: Les données ont été extraites de la base de données cliniques utilisée pour les soins directs aux patients (dossiers médicaux informatisés du programme de soin des maladies rénales chroniques), gérée prospectivement par le personnel soignant. La distance parcourue par le patient pour obtenir des soins a été estimée par un analyste du système d'informations géographiques de la Direction générale de la santé des Premières Nations et des Inuits, de Santé Canada. RÉSULTATS: Comparativement aux patients allochtones, les patients autochtones : présentaient une plus grande prévalence d'IRT (33,0 % vs 21,4 %; p < 0,001); présentaient un déclenchement plus précoce de la maladie (âge moyenAUT : 56,4 ans [SD=15,1]; âge moyenALL : 70,6 ans [SD=14,7]; p < 0,001) et un taux plus élevé d'IRT consécutive à un diabète de type 2 (66,1 % vs 39,0 %; p < 0,001); étaient plus susceptibles d'être dialysés (69,7 % vs 40,2 %; p < 0,001); recouraient moins à des traitements à domicile (AUT : 16,2 %; ALL : 25,7 %; p = 0,003); et étaient contraints de se déplacer davantage pour suivre leurs traitements (p < 0,001) ­ notamment, les autochtones étaient plus susceptibles de devoir parcourir au-delà de 200 km pour obtenir des soins. LIMITES: Les patients qui recevaient leurs traitements chez leur fournisseur de soins primaires ou uniquement via le cabinet de leur néphrologue n'étaient pas inclus dans l'étude. Les patients s'identifiant comme autochtones, mais ne possédant pas de numéros de CSI, n'ont pu être répertoriés aux fins de l'étude. CONCLUSION: En Saskatchewan, le fardeau différentiel que représente l'IRC chez les gens issus des Premières Nations se traduit par une atteinte plus sévère, par un moindre recours aux traitements à domicile et par de plus grandes distances à parcourir pour obtenir des soins. Des recherches supplémentaires sont requises pour proposer des solutions innovantes aux communautés partenaires des Premières Nations.

Primary pulmonary synovial sarcoma: a case report with unique and impressive computed tomography findings.

Primary pulmonary synovial sarcoma (PPSS) is a rare malignancy. Its etiology, imaging features and optimal treatment are not well understood. Pulmonary pseudoaneurysms and lymphadenopathy are rare complications of synovial sarcomas. A 40-year-old woman with mild hemoptysis and thoracic back pain underwent a computed tomography scan that revealed multiple pulmonary lesions, paraesophageal lymphadenopathy and incidental bilateral pulmonary emboli. A diagnosis of PPSS was made through the identification of an SS18 translocation by fluorescence in situ hybridization. She was started on adriamycin, ifosfamide and mesna chemotherapy. Over the subsequent two months, she developed three pulmonary artery pseudoaneurysms, ultimately requiring endovascular coiling. Seven months after starting treatment, the patient was asymptomatic. The lesions and lymphadenopathy decreased in size. The present case highlights complications of a rare malignancy and demonstrates positive response to ifosfamide-based chemotherapy in the setting of PPSS.

The (99m)Tc-DTPA urinary clearance method may be preferable to the plasma disappearance method for assessing glomerular filtration rate in diabetic nephropathy.

BACKGROUND: Isotopic glomerular filtration rate (iGFR) measurement is comparable to the inulin method. In this study, we compared urinary and plasma iGFR methodologies in patients with diabetic nephropathy. METHODS: A total of 147 patients from 3 sites in the Diabetic Intervention with Vitamins to Improve Nephropathy (DIVINe) trial provided 213 sets of urine and blood collections, at baseline, 18 and 36 months. RESULTS: The mean (with standard deviation) plasma iGFR of 60.7 (24.9) ml/min/1.73 m(2) compared to urinary iGFR of 52.0 (28.0) ml/min/1.73 m(2) was statistically significant (p value <0.001). Although plasma and urinary iGFRs were highly related (R(2) = 0.86), plasma iGFR increasingly overestimated urinary iGFRs at lower GFRs. In contrast to the cross-sectional analyses, the two measures of iGFR were weakly related (R(2) = 0.32) in regard to patients' change over 18 months of follow-up. CONCLUSION: Plasma iGFR may not be a suitable method for accurately measuring GFR in patients with advancing degrees of chronic kidney disease from diabetic nephropathy.

Epidemiology of acute kidney injury in Canadian critical care units: a prospective cohort study.

PURPOSE: We undertook this study to characterize the epidemiology of acute kidney injury (AKI) in Canadian critical care units. We aimed to identify predictors of mortality for patients diagnosed with AKI. METHODS: We conducted a prospective cohort study of consecutive patients admitted to critical care units at five Canadian hospitals over a 30-day period. Each patient was followed until hospital discharge or for a maximum of 30 days. The serum creatinine criteria for the Acute Kidney Injury Network (AKIN-SCr) system were used to identify, classify, and characterize patients who developed AKI. We used multivariable logistic regression to predict 30-day mortality among patients with AKI. RESULTS: We identified 603 patients, 161 (26.7%) of whom developed AKI. Compared to patients without AKI, those with AKI were more likely to die (29.2% vs 8.6%, P < 0.001). The risk of death increased with increasing AKIN-SCr stage (P < 0.001). In all, 19 patients (11.8% of those with AKI) commenced dialysis a median of one day (interquartile range, one to two days) after AKI diagnosis. At AKI diagnosis, the blood urea nitrogen (BUN) level (adjusted odds ratio [OR] 1.68, 95% confidence interval [CI] 1.01 to 2.79/10 mmol·L(-1)) and serum bicarbonate (adjusted OR 0.88, 95% CI 0.81 to 0.95/1 mmol·L(-1)) were associated with 30-day mortality and predicted death with an area under the receiver-operating characteristic curve of 0.79 (95% CI 0.71 to 0.86). CONCLUSIONS: Acute kidney injury is a common complication of critical illness in Canada. The development of even the mildest stage of AKI is associated with a substantially higher risk of death. At AKI diagnosis, routine clinical data may be helpful for predicting adverse outcomes.

Ampicillin/penicillin-binding protein interactions as a model drug-target system to optimize affinity pull-down and mass spectrometric strategies for target and pathway identification.

The identification and validation of the targets of active compounds identified in cell-based assays is an important step in preclinical drug development. New analytical approaches that combine drug affinity pull-down assays with mass spectrometry (MS) could lead to the identification of new targets and druggable pathways. In this work, we investigate a drug-target system consisting of ampicillin- and penicillin-binding proteins (PBPs) to evaluate and compare different amino-reactive resins for the immobilization of the affinity compound and mass spectrometric methods to identify proteins from drug affinity pull-down assays. First, ampicillin was immobilized onto various amino-reactive resins, which were compared in the ampicillin-PBP model with respect to their nonspecific binding of proteins from an Escherichia coli membrane extract. Dynal M-270 magnetic beads were chosen to further study the system as a model for capturing and identifying the targets of ampicillin, PBPs that were specifically and covalently bound to the immobilized ampicillin. The PBPs were identified, after in situ digestion of proteins bound to ampicillin directly on the beads, by using either one-dimensional (1-D) or two-dimensional (2-D) liquid chromatography (LC) separation techniques followed by tandem mass spectrometry (MS/MS) analysis. Alternatively, an elution with N-lauroylsarcosine (sarcosyl) from the ampicillin beads followed by in situ digestion and 2-D LC-MS/MS analysis identified proteins potentially interacting noncovalently with the PBPs or the ampicillin. The in situ approach required only little time, resources, and sample for the analysis. The combination of drug affinity pull-down assays with in situ digestion and 2-D LC-MS/MS analysis is a useful tool in obtaining complex information about a primary drug target as well as its protein interactors.

Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics.

Activation of cyclic nucleotide dependent signaling pathways leads to relaxation of smooth muscle, alterations in the cytoskeleton of cultured cells, and increases in the phosphorylation of HSP20. To determine the effects of phosphorylated HSP20 on the actin cytoskeleton, phosphopeptide analogs of HSP20 were synthesized. These peptides contained 1) the amino acid sequence surrounding the phosphorylation site of HSP20, 2) a phosphoserine, and 3) a protein transduction domain. Treatment of Swiss 3T3 cells with phosphopeptide analogs of HSP20 led to loss of actin stress fibers and focal adhesion complexes as demonstrated by immunocytochemistry, interference reflection microscopy, and biochemical quantitation of globular-actin. Treatment with phosphopeptide analogs of HSP20 also led to dephosphorylation of the actin depolymerizing protein cofilin. Pull-down assays demonstrated that 14-3-3 proteins associated with phosphopeptide analogs of HSP20 (but not peptide analogs in which the serine was not phosphorylated). The binding of 14-3-3 protein to phosphopeptide analogs of HSP20 prevented the association of cofilin with 14-3-3. These data suggest that HSP20 may modulate actin cytoskeletal dynamics by competing with the actin depolymerizing protein cofilin for binding to the scaffolding protein 14-3-3. Interestingly, the entire protein was not needed for this effect, suggesting that the association is modulated by phosphopeptide motifs of HSP20. These data also suggest the possibility that cyclic nucleotide dependent relaxation of smooth muscle may be mediated by a thin filament (actin) regulatory process. Finally, these data suggest that protein transduction can be used as a tool to elucidate the specific function of peptide motifs of proteins.

Average peptide score: a useful parameter for identification of proteins derived from database searches of liquid chromatography/tandem mass spectrometry data.

The quantity and variable quality of data that can be generated from liquid chromatography (LC)/mass spectrometry (MS)-based proteomics analyses creates many challenges in interpreting the spectra in terms of the actual proteins in a complex sample. In spite of improvements in algorithms that match putative peptide sequences to MS/MS spectra, the assembly of these lists of possible or probable peptides into a 'correct' set of proteins is still problematic. We have observed a trend in a simple relationship, derived from standard database search outputs, which can be useful in assessing the quality of a MS/MS-based protein identification. Specifically, the ratio of the protein score and number of non-redundant peptides, or average peptide score (APS), can facilitate initial filtering of database search results in addition to providing a useful measure of confidence for the proteins identified. This parameter has been applied to results from the analysis of multi-protein complexes derived from pull-down experiments analyzed using a two-dimensional LC/MS/MS workflow. In particular, the complex list of protein identifications derived from a drug affinity pull-down with immobilized ampicillin and an E. coli lysate was greatly simplified by applying the APS as a filter, allowing for facile identification of the penicillin-binding proteins known to interact with ampicillin. Furthermore, an APS threshold can be used for any data sets derived from electrospray ionization (ESI)- or matrix-assisted laser desorption/ionization (MALDI)-MS/MS experiments and is also not specific to any database search program.

Development of an LC-MALDI method for the analysis of protein complexes.

In this study, a two-dimensional LC-MALDI-TOF/TOF method has been developed for analyzing protein complexes. In our hands, the method has proven to be an excellent strategy for the analysis of protein complexes isolated in pull-down experiments. This is in part because the preservation of the chromatographic separation on a MALDI target yields an "unlimited" amount of time to obtain MS/MS spectra, making it possible to probe more deeply into complex samples. A brief statistical analysis was performed on the data obtained from the LC-MALDI-TOF/TOF system in order to better understand peptide fragmentation patterns under high-energy collision conditions. These statistical analyses provided some insight into how to evaluate the quality and accuracy of the database search results derived from the TOF/TOF-based analysis. The potential of the method was demonstrated by the successful identification of all the known penicillin-binding proteins in E. coli isolated using a drug-based pull-down with ampicillin as the bait. The performance of the LC-MALDI-TOF/TOF system was compared with that of an equivalent 2D LC-ESI-MS/MS approach, in the analysis of a protein bait-based pull-down. Regardless of the number of peptides identified in the ESI versus MALDI approach, the two approaches were found to be complementary. When the data is merged at the peptide level, the combined result gives higher Mascot scores and an overall higher confidence in protein identification than with either approach alone.