ABSTRACT
In our efforts to improve the identification of phosphopeptides by MS we have used peptide IEF on IPG strips. Phosphopeptides derived from trypsin digests of single proteins as well as complex cellular protein mixtures can be enriched by IEF and recovered in excellent yields at the acidic end of an IPG strip. IPG peptide fractionation in combination with MS/MS analysis has allowed us to identify phosphopeptides from tryptic digests of a cellular protein extract.
Subject(s)
Isoelectric Focusing/methods , Phosphopeptides/isolation & purification , Proteins/chemistry , Animals , Cell Line, Tumor , Humans , Mice , Phosphorylation , Proteins/metabolism , Proton-Motive Force , Receptor, Insulin/analysis , Recombinant Fusion Proteins/analysis , Tandem Mass Spectrometry , Trypsin/metabolismABSTRACT
The identification of disease markers in tissues and body fluids requires an extensive and thorough analysis of its protein constituents. In our efforts to identify biomarkers for affective and neurological disorders we are pursuing several different strategies. On one hand we are using animal models that represent defined phenotypes characteristic for the respective disorder in humans. In addition, we are analyzing human specimens from carefully phenotyped patient groups. Several fractions representing different protein classes from human cerebrospinal fluid obtained by lumbar puncture are used for this purpose. Our biomarker identification efforts range from classical proteomics approaches such as two dimensional gel electrophoresis and mass spectrometry to phage display screens with cerebrospinal fluid antibodies.
Subject(s)
Brain Diseases/metabolism , Proteome/analysis , Animals , Antibodies/analysis , Biomarkers , Cerebrospinal Fluid Proteins/analysis , Cerebrospinal Fluid Proteins/immunology , Disease Models, Animal , Electrophoresis, Gel, Two-Dimensional , Humans , Mass Spectrometry , Peptide Mapping , ProteomicsABSTRACT
For >15 generations, CD1 mice have been selectively and bidirectionally bred for either high-anxiety-related behavior (HAB-M) or low-anxiety-related behavior (LAB-M) on the elevated plus-maze. Independent of gender, HAB-M were more anxious than LAB-M animals in a variety of additional tests, including those reflecting risk assessment behaviors and ultrasound vocalization, with unselected CD1 "normal" control (NAB-M) and cross-mated (CM-M) mice displaying intermediate behavioral scores in most cases. Furthermore, in both the forced-swim and tail-suspension tests, LAB-M animals showed lower scores of immobility than did HAB-M and NAB-M animals, indicative of a reduced depression-like behavior. Using proteomic and microarray analyses, glyoxalase-I was identified as a protein marker, which is consistently expressed to a higher extent in LAB-M than in HAB-M mice in several brain areas. The same phenotype-dependent difference was found in red blood cells with NAB-M and CM-M animals showing intermediate expression profiles of glyoxalase-I. Additional studies will examine whether glyoxalase-I has an impact beyond that of a biomarker to predict the genetic predisposition to anxiety- and depression-like behavior.
Subject(s)
Anxiety Disorders/enzymology , Lactoylglutathione Lyase/metabolism , Analysis of Variance , Animals , Animals, Newborn , Anti-Anxiety Agents/administration & dosage , Anxiety Disorders/diagnosis , Anxiety Disorders/drug therapy , Anxiety Disorders/genetics , Avoidance Learning/drug effects , Avoidance Learning/physiology , Behavior, Animal , Biomarkers/metabolism , Blotting, Western/methods , Brain/drug effects , Brain/metabolism , Breeding/methods , Diazepam/administration & dosage , Disease Models, Animal , Electrophoresis, Gel, Two-Dimensional/methods , Exploratory Behavior/drug effects , Exploratory Behavior/physiology , Female , Hindlimb Suspension/physiology , Lactoylglutathione Lyase/isolation & purification , Locomotion/genetics , Male , Mass Spectrometry/methods , Mice , Microarray Analysis/methods , Phenotype , Predictive Value of Tests , Proteomics/methods , Reaction Time/physiology , Reproducibility of Results , Sex Factors , Spatial Behavior/drug effects , Spatial Behavior/physiology , Statistics as Topic , Swimming , Time Factors , Vocalization, Animal/drug effects , Vocalization, Animal/physiologyABSTRACT
We have analyzed the proteome of human cerebrospinal fluid with the help of shotgun mass spectrometry. In order to identify low-abundant proteins in these fluids, we have found it necessary to remove the abundant protein components from the mixture. Immunodepletion of the abundant proteins has allowed us to identify more than 100 proteins in cerebrospinal fluids from a patient suffering from normal pressure hydrocephalus. The identified proteins belong to a variety of different classes ranging from serum proteins to intracellular mediators that are involved in signal transduction and transcription. This work establishes a platform for future studies aimed at the comparative proteome analysis of cerebrospinal fluids from different groups of patients suffering from various psychiatric and neurological disorders.
