Li-Hua Lo

Acid hydrolysis followed by matrix-assisted laser desorption/ionization mass spectrometry for the rapid diagnosis of serum protein biomarkers in patients with major depression

We have developed a technique combining acid hydrolysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid study of the changes in the levels of positive and negative acute phase protein biomarkers in the sera of patients suffering from major depression. The serum proteins were first precipitated using an organic solvent; after separation, they were subjected to hydrochloric acid hydrolysis (6 M HCl) for 10 min. The resulting peptides were characterized using MALDI-TOF MS. Short-term treatment of the serum proteins with HCl efficiently removed interference from the abundant protein - albumin - and produced abundant peptide ion signals in the range of m/z 4000-10 000. This approach allowed us to rapidly detect the peptide ions originating from transferrin (a common negative acute phase protein) and fibrinogen (a common positive acute phase protein). The average ratios and (standard deviations) of the ion signals derived from transferrin/fibrinogen were 3.58 (+/-1.93) for the healthy control subjects and 1.02 (+/-0.52) for the patients suffering from major depression. The differences in transferrin/fibrinogen ratios between healthy controls and patients suggest that major depression will induce internal inflammation and cause either an increase in the level of fibrinogen or a decrease in the level of transferrin.

Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to rapidly screen for albuminuria.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is used as an alternative method for the rapid diagnosis of albuminuria. This technique requires no further sample pretreatment than simply mixing the urine sample with a MALDI matrix and drying under ambient conditions. The resulting MALDI mass spectra reveal albumin ions having charges ranging from +1 to +5. The detection of albumin is possible using any of the three most common MALDI matrices - sinapinic acid (SA), 2,5-dihydroxybenzoic acid (2,5-DHB), or 4-hydroxy-alpha-cyanocinnamic acid (alpha-CHC). Using this analytical approach, the limit of detection for albumin in urine is 10(-6) M, approximately 5 to 10 times lower than that detectable through conventional chemical testing.

Serum free hemoglobin as a novel potential biomarker for acute ischemic stroke

ObjectiveCurrently, no practical biomarker is available for the diagnosis of acute ischemic stroke. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has being applied extensively to analyze large biomolecules such as proteins. The technique is likely to be of remarkable value as indicators of systemic processes such as atherosclerosis and stroke. The aim of this study is to identify potential protein biomarkers for ischemic stroke diagnosis utilizing MALDITOF MS.MethodsSerum samples obtained from acute ischemic stroke patients (n = 47) and controls (n = 34) were analyzed by MALDI-TOF MS. Stroke severity was assessed by the National Institutes of Health Stroke Scale (NIHSS), subtype by the Trial of Org 10172 in Acute Stroke Treatment (TOAST), functional outcome by the modified Rankin Scale (mRS) and infarct volume by the diffusion-weighted images. Risk factors and routine laboratory data of the stroke patients were registered prospectively.ResultsThe peaks of hemoglobin (Hb) α-chain and β-chain were differentially expressed between stroke patients and controls (p < 0.0001). Hb ions were detected in the samples collected from 33 (70.2 %) stroke patients and 5 (14.7 %) controls. The sensitivity is 70.2 % and the specificity is 85.3 %. Among stroke patients, there is no significant correlation (p > 0.05) between Hb peaks and the NIHSS, TOAST, mRS, stroke risk factors, infarct volume, infarct location and laboratory data.ConclusionsSerum free Hb may serve as a novel potential biomarker for the diagnosis of acute ischemic stroke. The clinical value of this potential biomarker may be clarified by further studies quantifying serum free Hb levels.

Attenuated glial reaction in experimental proliferative vitreoretinopathy treated with liposomal doxorubicin.

PURPOSE We investigated the therapeutic effect of liposomal doxorubicin (Lipo-dox) on experimental proliferative vitreoretinopathy (PVR). METHODS The toxicity of Lipo-dox was determined in vitro in cultured rabbit retinal pigment epithelium (RPE) cells by tetrazolium-based (MTT) assay for cell viability performed 48 and 96 hours after treatment, and in vivo by electroretinography and histopathology. The therapeutic effect of intravitreous injection of Lipo-dox was evaluated in a rabbit model of PVR induced by injection of rabbit RPE cells after gas compression of the vitreous. The presence of PVR was determined by indirect ophthalmoscopy on days 1, 7, 14, 21, and 28 after injection. Western blot and immunofluorescence studies were performed to evaluate the expression of the glial markers vimentin and glial fibrillary acidic protein (GFAP). A pharmacokinetic study also was performed and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS). RESULTS The 50% inhibitory concentrations (IC₅₀) of doxorubicin (Doxo) and Lipo-dox in RPE cells were 0.01-0.1 and 0.1-1.0 μg/mL, respectively. Lipo-dox (10 μg/mL) did not reduce the amplitude reduction in the ERG study or produce obvious retinal toxicity. Lipo-dox still could be detected in the vitreous 7 days after injection. The Lipo-dox (10 μg/mL)-treated eyes showed lower grade PVR than did the untreated eyes. Lipo-dox also decreased the retinal expression levels of vimentin and GFAP. CONCLUSIONS Lipo-dox can attenuate the severity of experimental PVR, and reduces the glial cell expression of intermediate filaments in PVR retinas. Lipo-dox has a wider safe dosage range and a longer half-life in the vitreous than does primary Doxo.

Study of human neutrophil peptides in saliva by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is used to rapidly characterize the human neutrophil peptides - HNP 1, 2, and 3 - in saliva. The saliva excreted from the parotid and sublingual/submandibular glands of 70 individuals were collected and examined using MALDI-TOF. The MALDI approach requires no sample pretreatment other than mixing the saliva-absorbing material with the matrix and drying under ambient conditions. Tissue paper was the best material for collecting the saliva samples because of its strong texture and high absorbance, and sinapinic acid was the best MALDI matrix for the analysis of the HNPs. HNPs were detected in almost all the samples collected from the parotid glands, with no obvious differences among age or gender. In contrast, the distribution of the HNPs in the samples collected from the sublingual/submandibular glands was age-dependent: no HNPs were detected for those collected from individuals younger than 30, but the HNPs were present in all of the samples collected from those older than 60 years. The increased probability of detecting saliva HNPs with age suggests that HNPs may function as a biomarker for aging.

Characterization of human neutrophil peptides (α-Defensins) in the tears of dry eye patients

The potential peptide biomarkers of dry eye disease were rapidly and sensitively screened in tears by simple sample pretreatment and modern mass spectrometry. The tear sample was collected by simply wiping the corner of the eye with a small piece of Schirmer filter paper from the patients confirmed to have dry eye disease and from normal controls. The peptides adsorbed on the filter paper were extracted by vortexing it with sinapinic acid solution in a vial. A drop of the extracted solution was then deposited on the sample plate and examined using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS) without any other sample pretreatment. We found human neutrophil peptides (HNPs), so called α-defensins 1–3, which are potential biomarkers for inflammation, were significantly up-regulated in the tears collected from all the dry eye disease patients. The identity of the α-defensins in human tears was confirmed through (i) matching their molecular ions with those of standards, (ii) literature searches and (iii) electrophoretic purification and separation of the tears followed by in-gel tryptic digestion and MALDI-TOF analysis. The presence of large quantities of HNPs in the tears of dry eye patients suggests the occurrence of chronic inflammation in the ocular regions of dry eye patients.

The study of the distribution of melamine in rat renal tissues by imaging mass spectrometry

The recent tainting events of milk and pet foods with melamine and melamine-related compounds (MRCs), in attempts to elevate the total nitrogen content, have led to many cases of acute renal failure in infants and domestic pets. In this study, we employed matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry-based imaging mass spectrometry (IMS) technique to visualize the distribution of melamine in renal tissue. For rats orally administered with melamine alone, the protonated melamine ion at m/z 127 was detected only in the papilla. No melamine signal was detected in the kidney of rats receiving cyanuric acid alone. Melamine was accumulated in kidney only when the rats received high dose of melamine (100 mg day−1) together with cyanuric acid for 3 days. Under such regimen, the molecular signal was visible in both cortex and medulla, with an accumulation in renal papilla. Our IMS results for rat renal tissue are consistent with previous reports and histological observations. Nonetheless, there was no difference between the lipid distributions on the renal tissues of the control rats and those administered with melamine/cyanuric acid.

Detection of α–defensin in blister fluids as potential biomarkers for bullous pemphigoid patients by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry

BACKGROUND Bullous pemphigoid (BP) is a chronic blistering disease that manifests as multiple tense bullae on the limbs and body. Detecting biomarkers present in skin fluids may assist in the early diagnosis and treatment of BP. In this study, a modern mass spectrometric method was developed for screening biomarkers in blister fluids collected from patients. METHODS Blister fluids collected from BP patients and physically injured patients were analyzed and compared using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The blister fluids were mixed with MALDI matrix solution on the target plate; after drying, they were analyzed by MALDI-TOF MS. RESULTS Alpha-defensins 1-3 were detected in the samples collected from all BP patients and absent in all patients with physical injuries. Therefore, alpha-defensins 1-3 are potential biomarkers for BP and can be used to differentiate between blisters caused by BP and those caused by physical injuries. Compared to traditional skin biopsy methods that use immunofluorescent stains, analyzing biomarkers in blister fluids using MALDI-TOF is a more rapid and less invasive method. CONCLUSIONS MALDI-TOF-MS is a non-invasive and efficient method that is able to rapidly distinguish between blisters caused by BP and those caused by physical injuries.