Late-Onset Leber's Hereditary Optic Neuropathy with Concurrent Retinal Detachment and Retrobulbar Visual Pathway Involvement.

We report a case of late-onset Leber's hereditary optic neuropathy (LHON) with concurrent retinal detachment, mild retinal pigment epithelial changes, cataract and hyperintensity on fluid-attenuated inversion recovery magnetic resonance imaging affecting the entire retrobulbar visual pathway. We also documented that progression of the visual field defect correlated with retinal nerve fibre layer and ganglion cell layer-inner plexiform layer changes on optical coherence tomography. Our case provides further understanding of LHON as a disorder of the entire pre-geniculate pathways and also highlights that detailed history taking in addition to recognition of typical sequential optic disc appearance and visual field characteristics at different stages of LHON remain critical even in this era of modern imaging, autoimmune biomarkers and genetic testing.

Lowering Barriers to Health Risk Assessments in Promoting Personalized Health Management.

This study investigates the feasibility of accurately predicting adverse health events without relying on costly data acquisition methods, such as laboratory tests, in the era of shifting healthcare paradigms towards community-based health promotion and personalized preventive healthcare through individual health risk assessments (HRAs). We assessed the incremental predictive value of four categories of predictor variables-demographic, lifestyle and family history, personal health device, and laboratory data-organized by data acquisition costs in the prediction of the risks of mortality and five chronic diseases. Machine learning methodologies were employed to develop risk prediction models, assess their predictive performance, and determine feature importance. Using data from the National Sample Cohort of the Korean National Health Insurance Service (NHIS), which includes eligibility, medical check-up, healthcare utilization, and mortality data from 2002 to 2019, our study involved 425,148 NHIS members who underwent medical check-ups between 2009 and 2012. Models using demographic, lifestyle, family history, and personal health device data, with or without laboratory data, showed comparable performance. A feature importance analysis in models excluding laboratory data highlighted modifiable lifestyle factors, which are a superior set of variables for developing health guidelines. Our findings support the practicality of precise HRAs using demographic, lifestyle, family history, and personal health device data. This approach addresses HRA barriers, particularly for healthy individuals, by eliminating the need for costly and inconvenient laboratory data collection, advancing accessible preventive health management strategies.

Protein profiling in human sera for identification of potential lung cancer biomarkers using antibody microarray.

To identify potential biomarkers of lung cancer (LC), profiling of proteins in sera obtained from healthy and LC patients was determined using an antibody microarray. Based on our previous study on mRNA expression profiles between patients with LC and healthy persons, 19 proteins of interest were selected as targets for fabrication of an antibody microarray. Antibody to each protein and five nonspecific control antibodies were spotted onto a hydrogel-coated glass slide and used for profiling of proteins in sera of LC patients in a two-color fluorescence assay. Forty-eight human sera samples were analyzed, and expression profiling of proteins were represented by the internally normalized ratio method. Six proteins were distinctly down-regulated in sera of LC patients; this observation was validated by Wilcoxon test, false discovery rate, and Western blotting. Blind test of other 32 human sera using the antibody microarray followed by hierarchical clustering analysis revealed an approximate sensitivity of 88%, specificity of 80%, and an accuracy of 84%, respectively, in classifying the sera, which supports the potential of the six identified proteins as biomarkers for the prognosis of lung cancer.

A Mobile-Based Comprehensive Weight Reduction Program for the Workplace (Health-On): Development and Pilot Study.

BACKGROUND: There is a growing interest in mobile technology for obesity management. Despite the known effectiveness of workplace-based weight loss programs, there are few studies on mobile phone-delivered interventions. OBJECTIVE: This study aimed to develop and verify an integrated and personalized mobile technology-based weight control program, named Health-On, optimized for workplaces. METHODS: A weight reduction algorithm was developed for calorie prescription, continuous monitoring, periodic feedback and reevaluation, goal resetting, and offline intervention with behavior-changing strategies. A total of 30 obese volunteers (body mass index ≥25 kg/m2) participated in the 12-week Health-On pilot program. The primary outcome was weight reduction, and secondary outcomes were improved anthropometric measures, metabolic profiles, and fat computed tomography measures, all assessed pre- and postintervention. RESULTS: Health-On incorporated proprietary algorithms and several strategies intended to maximize adherence, using compatible online and offline interventions. The mean weight of 30 participants decreased by 5.8%, and median weight also decreased from 81.3 kg (interquartile range [IQR] 77.1-87.8) before intervention to 76.6 kg (IQR 70.8-79.5) after the 12-week intervention period (P<.001). The metabolic profiles and fat measures (blood pressure, glycosylated hemoglobin, total cholesterol, triglyceride, high-density lipoprotein, low-density lipoprotein, alanine aminotransferase, and visceral and subcutaneous adipose tissue; P<.05) also improved significantly. CONCLUSIONS: In this single-group evaluation of 30 participants before and after the Health-On program, body weight decreased and metabolic profiles and fat measures improved. Follow-up studies are needed to assess effectiveness and long-term adherence.

Energy transfer-based multiplexed assay of proteases by using gold nanoparticle and quantum dot conjugates on a surface.

Rapid and sensitive assay of proteases and their inhibition in a high-throughput manner is of great significance in the diagnostic and pharmaceutical fields. We developed a multiplexed assay system of proteases and their inhibition by measuring the energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs) on a glass slide. In this system, while the photoluminescence (PL) of donor QDs immobilized on a surface was quenched due to the presence of AuNPs (energy acceptor) in close proximity, the protease activity caused modulation in the efficiency of the energy transfer between the acceptor and donor, thus enabling the protease assay. In comparison to the QD-dye system, the conjugate of the QD-AuNP gave rise to higher energy transfer efficiency, resulting in quantitative assay of proteases with more sensitivity. When matrix metalloproteinase, caspase, and thrombin were tested, a multiplexed assay was successfully achieved since the AuNP could be used as a common energy acceptor in conjunction with QDs having different colors. Our system is anticipated to find applications in the diagnosis of protease-related diseases and screening of potential drugs with high sensitivity in a high-throughput way.

Importance of Active Participation in Obesity Management Through Mobile Health Care Programs: Substudy of a Randomized Controlled Trial.

BACKGROUND: Due to the prevalence of the westernized dietary pattern and lack of physical activity, the numbers of overweight or obese individuals are increasing, resulting in a growing health burden because of various related diseases. A lifestyle modification approach has additional advantages compared with pharmacological therapies or bariatric surgery. In our randomized controlled trial conducted in 2015, we successfully used a ubiquitous health care (SmartCare) service for patients with metabolic syndrome to achieve a significant weight loss effect. Various useful apps have been developed for the SmartCare Service, which involves using a mobile phone to manage chronic diseases, minimizing time and space restrictions. Many studies have demonstrated weight loss effects using a SmartCare service, but limited data are available regarding the effect of active participation in relation to weight loss. OBJECTIVE: We aimed to assess the weight loss effect achieved after using the SmartCare service in terms of adherence and participation. We divided the intervention group of the previous study according to participation level, and analyzed whether there was a significant difference in the outcome. METHODS: We classified participants into 3 groups according to their adherence. Within the intervention group using the SmartCare service, the active group comprised those transmitting anthropometric measurement data using a mobile phone 3 or more times per week or who had a health consultation 5 or more times during a 24-week period. The passive group comprised those who did not adhere to these levels of engagement. The control group comprised those who did not use the SmartCare service. We compared changes in body weight, body mass index (BMI), body fat percentage, waist circumference, and lipid profile among the 3 groups. RESULTS: We identified 422 participants and analyzed 405, excluding 17 who were missing necessary data for analysis. The active group consisted of 116 participants, compared with 80 in the passive group and 209 in the control group (without SmartCare service). There was a statistically significant difference in improvements to body weight, BMI, body fat percentage, and waist circumference among active participants compared with less active participants and the control group (P<.05). However, the lipid profile changes did not differ significantly between groups. CONCLUSIONS: The level of participation may be related to improved weight-related outcomes, which may improve health outcomes. To maximize the effectiveness of the SmartCare service, encouraging active participation is important. TRIAL REGISTRATION: Clinicaltrials.gov NCT01344811; https://clinicaltrials.gov/ct2/show/NCT01344811 (Archived by WebCite at http://www.webcitation.org/6alT2MmIB).

Chip-based analysis of SUMO (small ubiquitin-like modifier) conjugation to a target protein.

A chip-based analysis of protein interactions and modifications in cell signaling pathways has been of great potential in drug discovery, diagnostics, and cell biology, because it enables rapid and high-throughput biological assays with a small amount of samples. We report a chip-based analysis of sumoylation, the post-translational modification (PTM) process that involves covalent attachment of the small ubiquitin-like modifier (SUMO) protein to a target protein through multiple enzyme reactions in eukaryotic cells. Substrate proteins were spotted onto a glass surface followed by the addition of the reaction mixture for sumoylation, and the SUMO conjugation was readily detected by using fluorescent dye-labeled antibody. Under the optimized condition, on-chip sumoylation of Ran GTPase-activating protein 1 (RanGAP1) domain resulted in highly specific fluorescence intensity compared to that of its mutant (K524A) irrelevant to SUMO conjugation. The on-chip sumoylation was also verified and quantified by using the surface plasmon resonance(SPR) spectroscopy. As the exemplary study for a parallel analysis of sumoylation, fluorescent detection of sumoylation was conducted in a microarray format on a glass slide. The chip-based analysis developed here is expected to be applicable to assay for screening of target proteins from existing protein pools and proteome arrays in a high throughput manner.

Optimization of critical factors affecting the performance of an allergen chip for the analysis of an allergen-specific human IgE in serum.

A sensitive and multiplexed assay of allergen-specific human immunoglobulin E (IgE) is of great significance in the precise diagnosis of allergies. We report on the optimization of critical factors for chip-based analysis of IgE in human serum with a high reliability. Extracts of two mite species were used as model allergens, and were spotted onto a glass slide for the construction of an allergen chip. Respective allergen-specific IgE in human serum was analyzed by using biotinylated anti-human IgE and a streptavidin-Cy3 conjugate. Factors affecting the performance of the allergen chip were investigated and optimized. Especially, the effect of additives, the concentrations of biotinylated anti-human IgE and the streptavidin-Cy3 conjugate, the serum dilution factor, and the concentration of allergen extract as a capturing agent were examined in detail. Under the optimized conditions, a chip-based analysis for sera from 43 patients revealed a reliable and reproducible diagnosis of respective allergies, showing a good correlation with a conventional CAP assay.

Performance on physical function tests and the risk of fractures and admissions: Findings from a national health screening of 557,648 community-dwelling older adults.

OBJECTIVE: Falls and fractures in older adults are often preventable, yet remain major health concerns as comprehensive physical function assessment may not be readily available. This study investigated whether simple timed up and go test (TUG) and unipedal stance test (UST) are effective in identifying people with an increased risk of fractures, femoral fractures, or admissions due to femoral fractures. METHODS: Community-dwelling Korean older adults aged 66 years participated in the Korean National Screening Program for the Transitional Ages (n=557,648) between 2007 and 2010. Overall fractures, femoral fractures, and admissions due to femoral fracture during this period were outcome measures. The outcome measures were overall fractures, femoral fractures, and admissions due to femoral fracture after the health screening. The associations between inferior physical function test results and outcome measures were evaluated. RESULTS: A total of 523,502 subjects were followed-up for a mean period of 1.42 years, which resulted in 12,965 subjects with any fractures. Fracture data were retrieved from medical claims record. Subjects who performed poorly on one or both of the two physical function tests experienced higher number of overall fractures (aHR 1.21, 95% CI: 1.16-1.26), femoral fractures (aHR 1.80, 95% CI: 1.59-2.17), and admissions due to femoral fractures (aHR 1.85, 95% CI: 1.55-2.22) as compared to subjects with normal results on both tests. Combining TUG and UST was not superior to performing UST alone in predicting the increased risk of overall fractures (p=0.347), femoral fractures (p=0.402) or admissions due to femoral fractures (p=0.774). CONCLUSIONS: Poor performance on physical performance tests is associated with a higher risk of overall fractures, femoral fractures and admissions due to femoral fractures. The TUG and UST can be used to identify community-dwelling older individuals who are more vulnerable to fractures.

The Effectiveness of Mobile Phone-Based Care for Weight Control in Metabolic Syndrome Patients: Randomized Controlled Trial.

BACKGROUND: Overweight and obesity, due to a Westernized diet and lack of exercise, are serious global problems that negatively affect not only personal health, but national economies as well. To solve these problems, preventative-based approaches should be taken rather than medical treatments after the occurrence of disease. The improvement of individual life habits, through continuous care, is thus a paramount, long-term treatment goal. This study describes the effects of ubiquitous health care (uHealth care) or SmartCare services in the treatment of weight loss and obesity. OBJECTIVE: The aim of this study is to evaluate the effect of SmartCare services on weight loss compared to the effects of existing outpatient treatments in obese patients with metabolic syndrome. METHODS: Metabolic syndrome patients who met the inclusion/exclusion criteria were enrolled in the study and randomized into an intervention or control group. The intervention group was provided with remote monitoring and health care services in addition to the existing treatment. The control group was provided with only the existing treatment. Pedometers were given to all of the patients. Additionally, mobile phones and body composition monitors were provided to the intervention group while body weight scales were provided to the control group. The patients visited the hospitals at 12 and 24 weeks following the baseline examination to receive efficacy and safety evaluations. RESULTS: Mean weight reduction from baseline to week 24 was measured as a primary efficacy evaluation parameter and was found to be 2.21 kg (SD 3.60) and 0.77 kg (SD 2.77) in the intervention and control group, respectively. The intervention group had a larger decrement compared to the control group (P<.001). Among the secondary efficacy evaluation parameters, body mass index (BMI) (P<.001), body fat rate (P=.001), decrement of waist measurement (P<.001), and diet habit (P=.012) improvement ratings from baseline to week 24 were found to be superior in the intervention group compared with the control group. The proportion of patients whose body weight decreased by ≥10%, lipid profiles, blood pressure, prevalence of metabolic syndrome, change in the number of metabolic syndrome elements, smoking rate, drinking rate, and physical activity were not statistically significant between the groups. CONCLUSIONS: The efficacy of SmartCare services was confirmed as the intervention group that received both SmartCare services and the existing treatment had superior results compared with the control group that only received the existing treatment. Importantly, no specific problems with respect to safety concerns were observed. SmartCare service is thus an effective way to control the weight of obese patients with metabolic syndrome. TRIAL REGISTRATION: Clinicaltrials.gov NCT01344811; https://clinicaltrials.gov/ct2/show/NCT01344811 (Archived by Webcite at http://www.webcitation.org/6alT2MmIB).

Perceived risk as a barrier to appropriate diagnosis of irritable bowel syndrome.

AIM: To evaluate perceived risk, diagnostic testing, and acceptance of a diagnosis of irritable bowel syndrome (IBS) among the Korean laypersons. METHODS: We designed a conceptual framework to evaluate the health-seeking behavior of subjects based on a knowledge, attitude, and practice model. We developed a vignette-based questionnaire about IBS based on a literature review and focused group interviews. The vignette described a 40-year-old woman who meets the Rome III criteria for IBS without red-flag signs. It was followed by questions about demographic characteristics, health behaviors, IBS symptoms, risk perception, perceived need for diagnostic tests, and acceptance of a positive diagnosis of IBS. We planned a nationwide survey targeting laypersons without IBS and between the ages of 20 and 69 years. Survey participants were selected by quota sampling stratified by gender, age, and nationwide location. A multivariate logistic model was constructed based on literature reviews, univariate analysis, and a stepwise selection method to investigate correlations between the perceived risk, need for diagnostic tests, and acceptance of a positive diagnosis. RESULTS: Of 2354 eligible households, 1000 subjects completed the survey and 983 subjects were analyzed, excluding those who met symptom criteria for IBS. After reading the IBS vignette, the majority of subjects (86.8%) responded that the patient was at increased risk of severe disease. The most frequent concern was colon cancer (59.8%), followed by surgical condition (51.5%). Most subjects responded the patient needs diagnostic tests (97.2%). Colonoscopy was the most commonly required test (79.5%). Less than half of the respondents requested a stool examination (45.0%), blood test (40.7%), abdominal ultrasound (36.0%), or computed tomography (20.2%). The subjects who felt increased risk were more likely to see a need for colonoscopy [adjusted odds ratio (aOR) = 2.10, 95%CI: 1.38-3.18]. When asked about the positive diagnosis, the most frequent response was that "the patient would not be reassured" (65.7%). The increased risk perception group was less likely to be reassured by a positive diagnosis of IBS, compared to the other respondents (aOR = 0.52, 95%CI: 0.34-0.78). CONCLUSION: For IBS diagnosis, increased risk perception is a possible barrier to the appropriate use of diagnostic tests and to the patient's acceptance of a positive diagnosis.

Non Surgical Treatment of Eagle's Syndrome - A Case Report -.

Eagle's syndrome is a disease without a clear lesion that is associated with repeated episodes of pharyngalgia, odynophagia, the sensation of a foreign body in the pharynx, tinnitus, and otalgia in which patients displaying these types of symptoms must be given a differential diagnosis. It is known to be characterized by styloid process elongation or increasing compression to adjacent anatomical structures through stylohyoid ligament calcification. In serious cases, continuous pressure to the carotid artery can lead to a stroke. Diagnosis is confirmed through clinical symptoms, radiological findings, and physical examinations. The most common type of treatment consists of a surgical excision of elongated styloid process. Nonetheless, this study presents a case of treating Eagle's syndrome with conservative management.

Bispectral index and their relation with consciousness of the patients who receive desflurane or sevoflurane anesthesia during wake-up test for spinal surgery for correction.

BACKGROUND: Wake-up tests may be necessary during surgery for kypho-scoliosis to ensure that spinal function remains intact. It is difficult to predict the time when patients can respond to a verbal command. We evaluated the effectiveness of the bispectral index (BIS) and its relation to patients' levels of consciousness in wake-up tests during desflurane and sevoflurane anesthesia. METHODS: Eighteen patients each were enrolled in the desflurane and sevoflurane groups for spinal correction surgery. We measured BIS values, blood pressure, heart rate, and consciousness state and time, at the points when patients responded during the wake-up test. RESULTS: The BIS values when patients made fists upon a verbal command (T3) were 86.7 ± 7.5 for desflurane and 90.3 ± 5.4 for sevoflurane. Patients in the desflurane group had significantly shorter wake up delays than those in the sevoflurane group (6.9 ± 1.8 min vs. 11.8 ± 3.6 min). However, there was no difference between the groups in the time between the response to a verbal command and the time when a patient moved their toes in response to verbal commands. No recall of the wake-up tests occurred in either group. CONCLUSIONS: The values obtained using the BIS index could to some extent predict the time of a patient's and would be informative during desflurane and sevoflurane anesthesia. Moreover, desflurane permitted faster responses to verbal commands than sevoflurane, and allowed the wake-up test to be performed sooner.

Magnetophoretic immunoassay of allergen-specific IgE in an enhanced magnetic field gradient.

We demonstrate a novel magnetophoretic immunoassay of allergen-specific immunoglobulin E (IgE) based on the magnetophoretic deflection velocity of a microbead that is proportional to the associated magnetic nanoparticles under enhanced magnetic field gradient in a microchannel. In this detection scheme, two types of house dust mites, Dermatophagoides farinae (D. farinae) and Dermatophagoides pteronyssinus (D. pteronyssinus), were used as the model allergens. Polystyrene microbeads were conjugated with each of the mite extracts followed by incubation with serum samples. The resulting mixture was then reacted with magnetic nanoparticle-conjugated anti-human IgE for detection of allergen-specific IgE by using sandwich immuno-reactions. A ferromagnetic microstructure combined with a permanent magnet was employed to increase the magnetic field gradient ( approximately 10(4) T/m) in a microfluidic device. The magnetophoretic velocities of microbeads were measured in a microchannel under applied magnetic field, and the averaged velocity was well correlated with the concentration of allergen-specific IgE in serum. From the analysis of pooled sera obtained from 44 patients, the detection limits of the allergen-specific human IgEs for D. farinae and D. pteronyssinus were determined to be 565 (0.045 IU/mL) and 268 fM (0.021 IU/mL), respectively. These values are 1 order of magnitude lower than those by a conventional CAP system. For evaluation of reproducibility and accuracy, unknown sera were subjected to a blind test by using the developed assay system, and they were compared with the CAP system. As a result, coefficient of variance was less than 10%, and the developed method enabled a fast assay with a tiny amount of serum ( approximately 10 microL).

Combined positron emission tomography/computed tomography for evaluation of presumed choroidal metastases.

BACKGROUND: Choroidal metastases are the most common intraocular malignancy and are the first sign of systemic malignancy in approximately one-third of patients. Of patients with no previous diagnosis of cancer, oncological evaluation fails to find the primary lesion in approximately 50% of cases. Newer imaging modalities such as combined positron emission tomography/computed tomography (PET/CT) may improve the yield of the systemic work-up. METHODS: Consecutive patients presenting with presumed choroidal metastases were evaluated with whole body combined PET/CT scanning. RESULTS: Four patients presenting to a tertiary referral hospital with choroidal metastases as the first sign of systemic malignancy were evaluated. In all four cases, PET/CT demonstrated the ocular lesion, and the primary malignancy which was confirmed by tissue biopsy. False-negative results were seen in two cases of cerebral metastases. PET/CT demonstrated lesions not visible on CT or magnetic resonance imaging in two cases. CONCLUSIONS: Combined PET/CT is a useful addition to the work-up of patients with choroidal metastases. It provides the opportunity to detect lesions not visible with other imaging modalities and the ability to image patients with contraindications to magnetic resonance imaging. It is essential to correlate PET images with clinical information and the results of other imaging modalities and tissue biopsy remains the gold standard in the diagnosis of malignancy. False positives and negatives can occur with PET/CT, and further research is needed before this promising technology becomes a routine part of the evaluation of patients with choroidal metastases.

Expression profiling of proteins in L-threonine biosynthetic pathway of Escherichia coli by using antibody microarray.

We demonstrate the use of an antibody (Ab) microarray for a comparative expression profiling of proteins in an L-threonine biosynthetic pathway of Escherichia coli between a parental strain (W3110) and L-threonine overproducing mutant (TF5015). On the basis of a global comparative transcriptome analysis between the two strains, 28 analytical target proteins were selected and subjected to a production of polyclonal Abs against them. An Ab microarray was constructed by spotting a set of produced antibodies on a glass slide, and was employed for a comparative expression profiling of the proteins between the two strains by a two-color fluorescence assay method. The performance of the Ab microarray was evaluated with respect to cross-reactivity of the antibodies, dye-labeling efficiency, and the nature of antigenic proteins. Of these, the cross-reactivity of the used antibodies was found to mainly cause the deviation of the observed expression ratios from the expected ones. To offset the deviations, correction factors were derived from a statistical analysis and introduced. As a result, ten proteins were categorized to be up-regulated, while one was down-regulated in TF5015. Expression profiling of proteins using the Ab microarray was further verified by comparison with Western blotting and 2-DE.

Gold nanoparticle-enhanced secondary ion mass spectrometry imaging of peptides on self-assembled monolayers.

We demonstrate the use of gold nanoparticles (AuNPs) to enhance the secondary ion emission of peptides in time-of-flight secondary ion mass spectrometry (TOF-SIMS). The signal intensity of peptides adsorbed onto AuNPs was significantly increased when compared to that of self-assembled monolayers (SAMs). This gold nanoparticle-enhanced SIMS, termed NE-SIMS, enabled the sensitive detection of subtle modifications of peptides, such as phosphorylation. From a quantitative analysis of the amounts of adsorbed peptides and AuNPs on SAMs using quartz crystal microbalance and surface plasmon resonance spectroscopy, the ratio of peptide molecule to AuNP on amine-SAMs was revealed to be 18-19:1. When considering the ratio of peptide to matrix (1:10(3)-10(6)) employed in a matrix-enhanced SIMS, the use of AuNPs gave rise to a significantly increased secondary ion emission of peptides. Peptides were adsorbed onto patterned AuNPs on SAMs using a microfluidic system, and well-contrasted molecular ion images were obtained. NE-SIMS is expected to be applied to a chip-based analysis of modification of biomolecules in a label-free manner.

Mass spectrometric analysis of affinity-captured proteins on a dendrimer-based immunosensing surface: investigation of on-chip proteolytic digestion.

The monolayer of fourth-generation poly(amidoamine) dendrimers was adopted to construct the immunoaffinity surface of an antibody layer. The antibody layer as a bait on the dendrimer monolayer was found to result in high binding capacity of antigenic proteins and a reliable detection. The affinity-captured protein at the immunosensing surface was subjected to direct on-chip tryptic digestion, and the resulting proteolytic peptides were analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The performance of the on-chip digestion procedure was investigated with respect to the ratio of trypsin to protein, digestion time, composition of a reaction buffer, and the amount of affinity-captured protein on a surface. Addition of a water-miscible organic solvent to a reaction buffer had no significant effect on the digestion efficiency under the optimized digestion conditions. The on-chip digestion method identified the affinity-captured bovine serum albumin (BSA), lysozyme, and ferritin at the level of around 100 fmol. Interestingly, the detected number of peptide hits through the on-chip digestion was almost similar regardless of the amount of captured protein ranging from low- to high-femtomole levels, whereas the efficiency of in-solution digestion decreased significantly as the amount of protein decreased to low-femtomole levels. The structural alignment of the peptide fragments from on-chip-digested BSA revealed that the limited exterior of the captured protein is subjected to attack by trypsin. The established detection procedures enabled the identification of BSA in the biological mixtures at the level of 0.1 ng/mL. The use of antibodies against the proteins involved in the metabolic pathway of L-threonine in Escherichia coli also led to discrimination of the respective target proteins from cell lysates.