Characterization Of Proteins and Other Molecules By Mass Spectrometry

点击翻译按钮获取中文摘要

Protein Characterization in Niemann-Pick Disease-Type C (NPC). Differentially expressed protein spots (R&#8805;1.20, p<0.05 and R&#8804;0.83, p<0.05) were isolated from Npc1 mutant female mice and littermate controls at 1, 3 and 5 weeks of age, and using mass spectrometric techniques, we have candidate identifications to date on more than 200 pairs of protein spots. Functional assays of a number of candidates with potential involvement with liposomal storage diseases and neuronal degeneration is continuing on the proteins identified. To date two candidates of particular interest are transthyretin and fatty acid binding protein. Although these protein identifications were made from mouse brain, both of these are intriguing potential biomarkers in human CSF. One reason for this is that decreased levels of cerebral spinal fluid transthyretin correlate with disease severity in Alzheimer disease and elevated levels of fatty acid binding protein have been determined in CSF of NPC patients. Cerebral Spinal Fluid Profiling (CSF) in NPC. Initial experiments have demonstrated that we can detect significant differences between cerebral spinal fluid from NPC patients and adult controls using our novel analytical approach to compare protein profiles using MALDI TOF (Matrix Assisted Laser Desorption Ionization-Time Of Flight) mass spectrometry. CSF samples have been collected from 37 well-characterized NPC patients. Currently the collection for NPC patients includes longitudinal samples collected over 2.5 years and five pre/post miglustat pairs. In addition a collection of pediatric CSF samples is in progress at Childrens National Medical Center. In addition to the use of our Analysis Of VAriance coupled with Principal Component Analysis (ANOVA-PCA) analytical approach to MALDI-TOF profiling, we have begun the process of confirming proteins found in these same CSF samples through the use of shotgun proteomics methodology, albeit on less complex systems than those to which this approach is generally applied. The proteins in the CSF will be quantified once digested for shotgun analysis using isotopically coded tags, iTRAQ. Lipid Quantification in Serum. We have continued developing methodology to quantify cardiolipins in human serum. This effort is in association with a clinical study underway in the Institute to evaluate the effects of antibiotic treatment of pregnant women colonized with the Group B streptococcal (GBS) organism. The hypothesis of the study is that the typical peri-natal penicillin treatment gives rise to a large increase of circulating cardiolipins in the infant which then leads to respiratory distress. It has been demonstrated in other studies in newborn sheep that the GBS organisms secrete a specific cell wall membrane cardiolipin with penicillin treatment and that this substance causes respiratory distress at levels corresponding to the injection of about 100 pmole/mL in serum; note that this concentration falls rapidly with a half life of a few minutes. It is not known whether the respiratory distress observed in a fraction of infants born to GBS colonized mothers is a result of a similar effect, or perhaps by a related effect caused by a release of endogenous cardiolipins stimulated by the bacterial death. The analytical approach involves the addition of an internal standard to a 200 uL serum sample, extractions by a combination of liquid-liquid and solid phase, followed by an LC-MS analysis that incorporates an extraction/recovery standard to monitor system quality control. We have shown that cardiolipin can be extracted from serum with approximately 90% efficiency. We have further shown that normal adult levels of (18:2)-4 cardiolipins are present in serum at levels < 10 fmole/uL, approximately 1000-fold lower than found by earlier, less accurate measurements. Recent measurements using a new instrument (sensitivity more than 100-fold greater and with mass accuracy <10 ppm for the singly charged ions) have shown consistently that (18:2)-4 levels in normal adult serum vary between 3 and 10 fmole/uL, with biological variations far greater than replicate measurements of an individual sample. In addition, other cardiolipins with saturated fatty acid moieties have been detected in several individuals. New Approach to Mass Spectrometric-Based Protein Identification. Current approaches to protein identification rely heavily on database matching of fragmentation spectra, while ignoring (in a scoring sense) the mass accuracy of the precursor ions. We have developed a method based originally upon MALDI TOF-TOF instrumentation that uses targeted peptide mass fingerprinting results to confirm MS/MS database search identifications. The method uses first-order spectral data that have heretofore been ignored by most search engines. In this method the distribution of mass errors of peptide matches in the first order spectrum is used to develop a probability model that is independent from MS/MS database search identifications. Peptide mass matches can come from both precursor ions that have been fragmented as well as those that are tentatively identified by accurate mass alone. This additional confirmation enables us to assign protein identifications to MS/MS based scores that are otherwise considered to be only of moderate quality. The probability model employed uses Bayesian Inference and the mass errors in the first-order spectra to assign peptide probabilities and the product of individual peptide probabilities yields the final protein scores. Results based on a commercially available standard mixture of intact proteins shows both very high confidence levels for the proteins found in a 1D gel separation of the mixture, but also has also demonstrated that so-called one hit wonders found in fragmentation experiments can be validated reliably using this method. We suggest that the method will be applicable to LC-MS/MS methodologies that have precursor mass ion accuracies of at least 100 ppm.