Prospective measurement of outcomes and complications of tibial tuberosity advancement using novel mini plates in small breed dogs.

Cranial cruciate ligament (CrCL) disease is a common orthopedic disease in canine patients. Tibial osteotomy procedures for the treatment of cranial cruciate ligament disease in small breed dogs (<15 kg) have previously been limited. A total of 22 client-owned dogs, 26 stifles, with cranial cruciate ligament disease were treated with novel mini-tibial tuberosity advancement plates. The most common intraoperative complications included the need for plate-cage overlap in 7 stifles (26.92%) and screw head fracture in 1 (3.85%). Post-operative complications included tibial tuberosity fracture (3.85%), post-operative medial patella luxation (7.69%), and persistent lameness (7.69%). Of the 26 stifles evaluated in the medium term (>6-12 months) post-operatively, 92.3% had no lameness, with the remaining 7.7% having Grade 1 lameness. A good to excellent clinical outcome was noted in all 26 stifles that underwent TTA with novel mini plates.

Assessing nocturnal scratch with actigraphy in atopic dermatitis patients.

Nocturnal scratch is one major factor leading to impaired quality of life in atopic dermatitis (AD) patients. Therefore, objectively quantifying nocturnal scratch events aids in assessing the disease state, treatment effect, and AD patients' quality of life. In this paper, we describe the use of actigraphy, highly predictive topological features, and a model-ensembling approach to develop an assessment of nocturnal scratch events by measuring scratch duration and intensity. Our assessment is tested in a clinical setting against the ground truth obtained from video recordings. The new approach addresses unmet challenges in existing studies, such as the lack of generalizability to real-world applications, the failure to capture finger scratches, and the limitations in the evaluation due to imbalanced data in the current literature. Furthermore, the performance evaluation shows agreement between derived digital endpoints and the video annotation ground truth, as well as patient-reported outcomes, which demonstrated the validity of the new assessment of nocturnal scratch.

Protein kinetics of superoxide dismutase-1 in familial and sporadic amyotrophic lateral sclerosis.

OBJECTIVE: Accumulation of misfolded superoxide dismutase-1 (SOD1) is a pathological hallmark of SOD1-related amyotrophic lateral sclerosis (ALS) and is observed in sporadic ALS where its role in pathogenesis is controversial. Understanding in vivo protein kinetics may clarify how SOD1 influences neurodegeneration and inform optimal dosing for therapies that lower SOD1 transcripts. METHODS: We employed stable isotope labeling paired with mass spectrometry to evaluate in vivo protein kinetics and concentration of soluble SOD1 in cerebrospinal fluid (CSF) of SOD1 mutation carriers, sporadic ALS participants and controls. A deaminated SOD1 peptide, SDGPVKV, that correlates with protein stability was also measured. RESULTS: In participants with heterozygous SOD1A5V mutations, known to cause rapidly progressive ALS, mutant SOD1 protein exhibited ~twofold faster turnover and ~ 16-fold lower concentration compared to wild-type SOD1 protein. SDGPVKV levels were increased in SOD1A5V carriers relative to controls. Thus, SOD1 mutations impact protein kinetics and stability. We applied this approach to sporadic ALS participants and found that SOD1 turnover, concentration, and SDGPVKV levels are not significantly different compared to controls. INTERPRETATION: These results highlight the ability of stable isotope labeling approaches and peptide deamidation to discern the influence of disease mutations on protein kinetics and stability and support implementation of this method to optimize clinical trial design of gene and molecular therapies for neurological disorders. TRIAL REGISTRATION: Clinicaltrials.gov: NCT03449212.

Test-Retest Instability of Temporal Summation and Conditioned Pain Modulation Measures in Older Adults.

OBJECTIVE: The temporal stability (test-retest reliability) of temporal summation of pain (TS) and conditioned pain modulation (CPM) has yet to be established in healthy older adults. The purpose of this study was to compare the temporal stability of TS and CPM in healthy older and younger adults and to investigate factors that might influence TS and CPM stability. METHODS: In a test-retest study, 40 healthy older adults and 30 healthy younger adults completed two sessions of quantitative sensory testing within a two-week period that included TS of heat pain, TS of mechanical pain, and CPM with pressure pain thresholds and suprathreshold heat pain as test stimuli and a cold water immersion as a conditioning stimulus. Participants also completed self-report measures of situational catastrophizing, anxiety, clinical pain, and physical activity. Absolute and relative stability were examined for each variable. Bivariate correlations examined the associations of age, clinical, behavioral, and psychological variables with the intra-individual stability of TS and CPM. RESULTS: The results revealed moderate to excellent stability for the TS measures and poor to moderate stability for CPM. The results also revealed significant age differences for two of the TS measures and CPM, with younger adults having greater stability compared with older adults. Additionally, the magnitude and stability of psychological factors were correlated with stability of TS. CONCLUSIONS: These findings suggest that TS and CPM may be more reliable in younger compared with older adults. Furthermore, psychological states may be an important factor influencing the stability of TS in healthy adults.

Assessing the Financial Toxicity of Radiation Oncology Patients Using the Validated Comprehensive Score for Financial Toxicity as a Patient-Reported Outcome.

PURPOSE: The financial burden of cancer care may significantly affect patient quality of life and clinical outcomes. However, the financial effect of radiation therapy on patients remains difficult to characterize, in part owing to the lack of standardized methods to measure patient distress related to treatment costs. Here, we assessed financial burden in the radiation oncology population by applying the Comprehensive Score for Financial Toxicity (COST), a patient-reported outcome measure, which has been validated in medical oncology patients.   METHODS AND MATERIALS: Consecutive patients from a single academic radiation oncology clinic were recruited. Participants completed the 11-item COST-Functional Assessment of Chronic Illness Therapy questionnaire, with total possible scores ranging from 0 to 44. Scores were collected along with data regarding patient demographics, insurance, diagnosis, and treatment. Univariate and multivariate analyses were performed to identify factors associated with higher financial burden as measured by COST. RESULTS: A total of 167 patients completed the COST questionnaire. Lower COST scores indicated higher financial toxicity. The population's mean COST score was 21.9 (95% confidence interval, 20.5-23.3). Fifteen percent of participants reported grade 2 to 3 COST toxicity, corresponding to a moderate or severe effect on quality of life. Use of concurrent or previous systemic therapy was significantly associated with lower COST scores on univariate analysis (P = .03), but not significant on multivariate analysis. A subset analysis of posttreatment follow-up patients identified rural residence and recent completion of radiation therapy as significant correlates of worse COST scores on univariate analysis, and rural residence remained independently associated on multivariate analysis (P = .017). CONCLUSIONS:  COST effectively identified a significant number of radiation oncology patients experiencing financial toxicity, indicating its prevalence in this population. A correlate of financial toxicity in this population is the use of systemic therapy. Of those who have completed radiation therapy, rural residence was independently associated with worse financial toxicity.

Loss of appetite in amyotrophic lateral sclerosis is associated with weight loss and decreased calorie consumption independent of dysphagia.

BACKGROUND: Loss of appetite has been reported to affect up to half of people with amyotrophic lateral sclerosis (ALS) and to be associated with weight loss. We wished to test whether loss of appetite correlates with reduced dietary intake independent of dysphagia. METHODS: Appetite was measured repeatedly using the Council on Nutrition Appetite Questionnaire (CNAQ) in participants in the Electronic health Application To Measure Outcomes REmotely study. Dietary intake and weight were compared to appetite, ALS Functional Rating Scale-Revised total and bulbar scores (dysphagia). RESULTS: The average baseline CNAQ score was 30.4 (n = 61; SD = 3.9) with 18.0% scoring <28 points (severe loss of appetite). Lower CNAQ scores correlated with greater weight loss since diagnosis (Pearson correlation coefficient, r = -0.34; P = 0.009) and lower baseline energy intake (P = 0.007), independent of dysphagia. CONCLUSIONS: Our results support an association between loss of appetite and decreased calorie intake and weight in ALS which is independent of dysphagia.

Opportunities for use of radiation therapy in penile cancer based on patterns of care in the United States from 2007 to 2013.

BACKGROUND: Radiation therapy (RT) is an effective modality for the treatment of squamous cell carcinomas of the penis. The National Comprehensive Cancer Network recommends consideration of primary radiation for penile preservation, in surgically unresectable tumors, and as adjuvant therapy for positive margins, bulky groin nodes or pelvic nodes. We performed a population-based analysis to evaluate the usage of RT in penile cancer from 2007 to 2013. METHODS: We used the Surveillance, Epidemiology and End Results (SEER) database to identify men diagnosed with squamous cell carcinoma of the penis from 2007 to 2013. Patients were grouped as early stage (T1-T2N0), locally advanced (T3-T4N0), node-positive (T1xN1-3) and metastatic. We used linear regression model to test for factors associated with adjuvant radiation in node-positive patients. RESULTS: We identified 2200 men diagnosed with penile cancer between 2007 and 2013. Of these, 66.4% had early stage, 10.7% had locally advanced, 15.5% had node-positive, 3.2% had metastatic cancer. Among patient with early stage cancer, RT was used in 14 patients (1.0%) and postoperative radiation in an additional 45 patients (3.1%). Among 340 patients with node-positive cancer, 62.1% received surgery alone, 5.6% radiation alone, 21.8% surgery with adjuvant radiation, and 10.6% neither surgery nor radiation. Of patients who had surgery, 26.0% had adjuvant radiation. On univariate analysis, higher nodal stage (N2-3 versus N1) was associated with adjuvant radiation (p = 0.02), while there was a trend for higher T-stage (T3/T4 versus T1/T2) (p = 0.08) and history of prior malignancy (p = 0.06). On multivariate analysis, only higher nodal stage (N2-3 versus N1) was associated with use of adjuvant radiation [hazard ratio (HR) 1.94, p = 0.03]. CONCLUSIONS: A small percentage of patient who are eligible for primary or adjuvant RT in the United States receive this treatment. Further work should be done to assess barriers to use of radiation in patients with penile cancer.

Physical Activity Levels Predict Exercise-induced Hypoalgesia in Older Adults.

Prior research indicates that older adults exhibit a deficient capacity to activate multiple pain inhibitory mechanisms, including pain inhibition after acute exercise termed exercise-induced hypoalgesia (EIH). The influence of physical activity levels and psychological processes on EIH in older adults remains unclear. PURPOSE: This study examined potential psychological and physical activity predictors of the magnitude of EIH after submaximal isometric exercise in healthy older adult men and women. METHODS: Fifty-two healthy older adults completed a test of EIH, the Pain Catastrophizing Scale, the Tampa Scale of Kinesiophobia, and wore an accelerometer on the hip for 1 wk to assess physical activity levels. For the test of EIH, participants complete a 3-min isometric handgrip at 25% of maximum voluntary contraction. Pressure pain thresholds (PPT) and a 30-s continuous heat pain test were completed before and immediately after the exercise. RESULTS: Mixed-model ANOVA revealed that older adults demonstrated significantly decreased PPT after isometric exercise (P = 0.030), and no changes on the heat pain trials from pretest to posttest (P > 0.05). A multiple regression revealed that accumulated moderate to vigorous physical activity (MVPA) per week significantly predicted the change in PPT after exercise (ß = 0.35, P = 0.012). Participants who averaged greater MVPA experienced a greater increase in PPT after exercise. No relationships were found with EIH and the psychological variables. CONCLUSIONS: Older adults did not exhibit EIH after submaximal isometric exercise. However, those who did more MVPA per week experienced a greater magnitude of pain inhibition after acute exercise.

Sensitivity to Physical Activity Predicts Daily Activity Among Pain-Free Older Adults.

Objective: Prior research indicates that older adults with knee osteoarthritis have increased sensitivity to physical activity (SPA) and respond to physical activities of stable intensity with increases in pain. Whether SPA is present in healthy older adults without chronic pain and predicts functional outcomes remains relatively unexplored. The purpose of this study was to determine the degree of SPA in healthy older adults in response to a standardized walking task, and whether SPA was associated with temporal summation of pain, pain-related fear of movement, and functional outcomes. Methods: Fifty-two older adults without chronic pain completed self-reported measures of activity-related pain and physical function, completed the Six-Minute Walk Test (6MWT), underwent quantitative sensory testing to measure temporal summation of heat pain, and wore an accelerometer for one week to measure physical activity behavior. Subjects rated overall bodily discomfort (0-100 scale) prior to and during each minute of the 6MWT. An SPA index was created by subtracting the initial bodily discomfort ratings from the peak ratings. Results: Repeated-measures analysis of variance indicated that bodily discomfort significantly increased across the walking task, with approximately 60% of the sample experiencing SPA. Hierarchical regressions indicated that greater SPA was associated with fewer average steps per day and greater activity-related pain. Additionally, analyses revealed that temporal summation of pain and pain-related fear of movement significantly predicted the degree of SPA on the walking task. Conclusions: These findings shed light on potential mechanisms underlying SPA in older adults and suggest that SPA might be a risk factor for reduced physical activity.

Nurse-midwives' ability to diagnose acute third- and fourth-degree obstetric lacerations in western Kenya.

BACKGROUND: Obstetric fistula devastates the lives of women and is found most commonly among the poor in resource-limited settings. Unrepaired third- and fourth-degree perineal lacerations have been shown to be the source of approximately one-third of the fistula burden in fistula camps in Kenya. In this study, we assessed potential barriers to accurate identification by Kenyan nurse-midwives of these complex perineal lacerations in postpartum women. METHODS: Nurse-midwife trainers from each of the seven sub-counties of Siaya County, Kenya were assessed in their ability to accurately identify obstetric lacerations and anatomical structures of the perineum, using a pictorial assessment tool. Referral pathways, follow-up mechanisms, and barriers to assessing obstetric lacerations were evaluated. RESULTS: Twenty-two nurse-midwife trainers were assessed. Four of the 22 (18.2%) reported ever receiving formal training on evaluating third- and fourth-degree obstetric lacerations, and 20 of 22 (91%) reported health-system challenges to adequately completing their examination of the perineum at delivery. Twenty-one percent of third- and fourth-degree obstetric lacerations in the pictorial assessment were incorrectly identified as first- or second-degree lacerations. CONCLUSION: County nurse-midwife trainers in Siaya, Kenya, experience inadequate training, equipment, staffing, time, and knowledge as barriers to adequate diagnosis and repair of third- and fourth-degree perineal tears.

Chronic restraint stress causes a delayed increase in responding for palatable food cues during forced abstinence via a dopamine D1-like receptor-mediated mechanism.

Relapse to unhealthy eating habits in dieters is often triggered by stress. Animal models, moreover, have confirmed a causal role for acute stress in relapse. The role of chronic stress in relapse vulnerability, however, has received relatively little attention. Therefore, in the present study, we used an abstinence-based relapse model in rats to test the hypothesis that exposure to chronic stress increases subsequent relapse vulnerability. Rats were trained to press a lever for highly palatable food reinforcers in daily 3-h sessions and then tested for food seeking (i.e., responding for food associated cues) both before and after an acute or chronic restraint stress procedure (3h/day×1day or 10days, respectively) or control procedure (unstressed). The second food seeking test was conducted either 1day or 7days after the last restraint. Because chronic stress causes dopamine D1-like receptor-mediated alterations in prefrontal cortex (a relapse node), we also assessed dopaminergic involvement by administering either SCH-23390 (10.0µg/kg; i.p.), a dopamine D1-like receptor antagonist, or vehicle prior to daily treatments. Results showed that chronically, but not acutely, stressed rats displayed increased food seeking 7days, but not 1day, after the last restraint. Importantly, SCH-23390 combined with chronic stress reversed this effect. These results suggest that drugs targeting D1-like receptors during chronic stress may help to prevent future relapse in dieters.

Considering sex as a biological variable in preclinical research.

In June 2015, the National Institutes of Health (NIH) released a Guide notice (NOT-OD-15-102) that highlighted the expectation of the NIH that the possible role of sex as a biologic variable be factored into research design, analyses, and reporting of vertebrate animal and human studies. Anticipating these guidelines, the NIH Office of Research on Women's Health, in October 2014, convened key stakeholders to discuss methods and techniques for integrating sex as a biologic variable in preclinical research. The workshop focused on practical methods, experimental design, and approaches to statistical analyses in the use of both male and female animals, cells, and tissues in preclinical research. Workshop participants also considered gender as a modifier of biology. This article builds on the workshop and is meant as a guide to preclinical investigators as they consider methods and techniques for inclusion of both sexes in preclinical research and is not intended to prescribe exhaustive/specific approaches for compliance with the new NIH policy.-Miller, L. R., Marks, C., Becker, J. B., Hurn, P. D., Chen, W.-J., Woodruff, T., McCarthy, M. M., Sohrabji, F., Schiebinger, L., Wetherington, C. L., Makris, S., Arnold, A. P., Einstein, G., Miller, V. M., Sandberg, K., Maier, S., Cornelison, T. L., Clayton, J. A. Considering sex as a biological variable in preclinical research.

The science of sex and gender in human health: online courses to create a foundation for sex and gender accountability in biomedical research and treatment.

BACKGROUND: Sex and gender differences play a significant role in the course and outcome of conditions that affect specific organ systems in the human body. Research on differences in the effects of medical intervention has helped scientists develop a number of sex- and gender-specific guidelines on the treatment and management of these conditions. An online series of courses, "The Science of Sex and Gender in Human Health," developed by the National Institutes of Health Office of Research on Women's Health and the U.S. Food and Drug Administration Office of Women's Health, examines sex and gender differences and their implications. Thus far, three online courses have been generated. The first course offers an overview of the scientific and biological basis for sex- and gender-related differences. The second course is focused on disease-specific sex and gender differences in health and behavior and their implications. Finally, the third course covers the influence of sex and gender on disease manifestation, treatment, and outcome. METHODS: Data were obtained using website analytics and post-course surveys. RESULTS: To date, over 1000 individuals have completed at least one course. Additionally, 600 users have received continuing education credit for completing a course in the series. Finally, the majority of respondents to the online course survey have indicated that the courses considerably enhanced their professional effectiveness. CONCLUSIONS: "The Science of Sex and Gender in Human Health" online courses are freely available sources of information that provide healthcare providers and researchers with the resources to successfully account for sex and gender in their medical practice and research programs.

A Report of the Women's Health Congress Workshop on The Health of Women of Color: A Critical Intersection at the Corner of Sex/Gender and Race/Ethnicity.

Women of color face unique health challenges that differ significantly from those of other women and men of color. To bring these issues to light, the National Institutes of Health (NIH) Office of Research on Women's Health sponsored a preconference workshop at the 23rd Annual Women's Health Congress, which was held in Washington, DC, in April 2015. The workshop featured presentations by NIH intramural and extramural scientists who provided insight on the disparities of a wide range of conditions, including cancer, cardiovascular disease, the risk of HIV infection, and disability in an aging population. In this study, we highlight the major points of each presentation and the ensuing discussion.

Effects of repeated yohimbine administration on reinstatement of palatable food seeking: involvement of dopamine D1 -like receptors and food-associated cues.

Acute exposure to the pharmacological stressor yohimbine induces relapse to both food and drug seeking in a rat model. However, no systematic studies on the effects of chronic stress on relapse have been conducted. Because chronic stress causes changes in dopamine D1 -like receptor-mediated transmission in prefrontal cortex (a relapse node), we tested the hypothesis that chronic exposure to stress increases vulnerability to relapse via dopamine-mediated mechanisms. Additionally, to determine the role of food-conditioned cues in reinstatement of food seeking, we made discrete food-paired cues either available (CS Present) or not available (CS Absent) during extinction and reinstatement testing. Rats responded for palatable food reinforcers in daily 3-hour sessions, and the behavior was extinguished. To model chronic stress, rats were injected daily with yohimbine (0.0, 2.5, or 5.0 mg/kg; i.p.) during the first 7 days of extinction. Injections were combined with SCH-23390 (0.0, 5.0, or 10.0 µg/kg; i.p.), a D1 -like receptor antagonist. Rats were then tested for reinstatement of food seeking triggered by acute yohimbine (0.0, 1.0, or 2.0 mg/kg; i.p.) and pellet priming. Rats treated previously with chronic yohimbine displayed increased responding following acute yohimbine priming relative to non-chronically stressed rats, but in the CS Absent condition only. Conversely, the lower dose of chronic yohimbine caused an increase in pellet-primed reinstatement, but this effect was more pronounced in the CS Present condition. Importantly, SCH-23390 combined with repeated yohimbine injections attenuated these effects. Thus, chronic stress may increase vulnerability to relapse under specific circumstances via a dopamine D1 -like receptor-mediated mechanism.

A label-free mass spectrometry method for relative quantitation of ß-tubulin isotype expression in human tumor tissue.

PURPOSE: Quantitation of ß-tubulin isotype expression in taxane resistant human tumor tissue has been difficult to achieve because of the limited availability of validated antibodies. Here we present a label-free MS method to quantitate relative expression levels of ß-tubulin isotypes. EXPERIMENTAL DESIGN: Using isotype-specific reporter peptides, we determined relative ß-tubulin isotype expression levels in human lung tumor tissue. RESULTS: Four reporter peptides were chosen to quantitate the ßI/ßII, ßIV, ßIII, and ßV tubulin isotypes. These peptides were validated using human cancer cell lines. The label-free method was then used to determine ß-tubulin isotype expression in nine human lung tumor samples, which had been described as high or low ßIII-tubulin expressing using immunohistochemistry. It was found that ßI/ßII (accounting for 18.7-65.7% of total ß-tubulin) and ßIVa/ßIVb (26.3-79.1%) were the most abundant isotypes and that the ßIII (0-8.9%) and ßV (1.0-10.4%) were less abundant in the tissue. We also categorized the samples as high or low ßIII-tubulin expressing. CONCLUSION AND CLINICAL RELEVANCE: With this method we can determine the relative expression levels of ß-tubulin isotypes in human tumor tissue. This method will facilitate studies assessing the use of tubulin isotypes as biomarkers of taxane resistance.

Frozen tissue can provide reproducible proteomic results of subcellular fractionation.

Differential detergent fractionation (DDF) is frequently used to partition fresh cells and tissues into distinct compartments. We have tested whether DDF can reproducibly extract and fractionate cellular protein components from frozen tissues. Frozen kidneys were sequentially extracted with three different buffer systems. Analysis of the three fractions with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1693 proteins, some of which were common to all fractions and others of which were unique to specific fractions. Normalized spectral index (SI(N)) values obtained from these data were compared to evaluate both the reproducibility of the method and the efficiency of enrichment. SI(N) values between replicate fractions demonstrated a high correlation, confirming the reproducibility of the method. Correlation coefficients across the three fractions were significantly lower than those for the replicates, supporting the capability of DDF to differentially fractionate proteins into separate compartments. Subcellular annotation of the proteins identified in each fraction demonstrated a significant enrichment of cytoplasmic, cell membrane, and nuclear proteins in the three respective buffer system fractions. We conclude that DDF can be applied to frozen tissue to generate reproducible proteome coverage discriminating subcellular compartments. This demonstrates the feasibility of analyzing cellular compartment-specific proteins in archived tissue samples with the simple DDF method.

Gel-based proteomics of liver cancer progression in rat.

A significant challenge in proteomics biomarker research is to identify the changes that are of highest diagnostic interest, among the many unspecific aberrations associated with disease burden and inflammation. In the present study liver tissue specimens (n=18) from six experimental stages were collected from the resistant hepatocyte (RH) rat model of liver cancer and analyzed by 2D DIGE. The study included triplicates of regenerating liver, control "sham-operated" liver, three distinct premalignant stages and hepatomas. Out of 81 identified proteins two-thirds were differentially abundant in rat hepatomas compared to control rat liver and, secondly, the majority of proteins were also changed in precursor stages. This underscores the importance of adequate control samples in explorative cancer biomarker research. We confirm several proteomic changes previously identified in human hepatocellular carcinoma (HCC) and we identify novel candidate proteomic aberrations for further analysis in human HCC. In particular, increased levels of HSP70, HSP90, AKR1B1, AKR7A3, GCLM, ANXA5, VDBP, RGN and SULT1E1 were associated specifically with rat hepatomas, or with liver cancer progression in rat. In addition, we examine an integrated gel-based workflow for analysis of protein post-translational modifications (PTMs) and microtubule-association. We highlight differential PTM and localization of HSP60 as an interesting target for further analysis in liver cancer.

Methods in tubulin proteomics.

New analytical methods are needed for the successful outcome of experiments aimed at characterizing mechanisms of microtubule dynamics and at understanding the effects of drugs on microtubules. The identification of tubulin isotypes and of regions of the microtubule involved in drug interactions has been advanced by proteomic methodologies. The diversity of tubulin sequences and posttranslational modifications (PTMs) can generate a complex mixture of heterodimers with unique molecular dynamics driving specific functions. Mass spectrometry (MS)-based approaches have been developed, and in combination with chromatographic and/or electrophoretic separation of tubulin polypeptides or peptides, they have contributed to our understanding of tubulin proteomics. We present protocols that we have used for the analysis of tubulin isotypes and PTMs present in tubulin isolated from cells in culture or tissues and for the identification of tubulin regions altered by microtubule-stabilizing agents. Tubulin proteomics complements structural and computer modeling information for a high-resolution view of microtubule dynamics and its alteration by drugs. These methodologies will help in providing insights into tubulin isotype-specific functions and in the design of drugs targeting either all tubulin heterodimers indiscriminately or only those containing specific isotypes.