Journal: The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians
Article Title: Isobaric labeling and tandem mass spectrometry: a novel approach for profiling and quantifying proteins differentially expressed in amniotic fluid in preterm labor with and without intra-amniotic infection/inflammation
doi: 10.3109/14767050903067386
Figure Lengend Snippet: The sequence of the workflow is indicated by the arrows in the right side. Pooled reference: Small aliquots from each of the 75 samples of amniotic fluid were pooled to create a reference sample. Deplete-concentrate-reduce-block: The amniotic fluid samples were depleted of albumin and IgG, subsequently concentrated and reduced. Digestion: The samples were subjected to trypsin digestion. Labeling: The reference sample and three individual samples (denoted A, B, and C in the figure) were labeled with iTRAQ reagents (the numbers 114–117 refer to the reporter mass of the individual iTRAQ reagent). Mixing and fractionation: The four labeled samples were mixed and subjected to strong cation exchange (SCX) fractionation to simplify the mixture prior to conducting liquid chromatography and mass spectrometry (LC-MS/MS). Each SCX fraction was subjected to LC-MS/MS. Liquid chromatography: Proteins were separated by liquid chromatography. The retention time is displayed in the horizontal axis. MS1: The first stage of mass spectrometry for a specific retention time is indicated schematically. Each peak in the mass spectrum (horizontal axis is m/z) corresponds to an individual peptide. MS2: A single peak is selected for collisional dissociation and the fragments subjected to a second stage mass spectrometry (tandem MS/MS). The reporter ions from the iTRAQ label appear in the low mass range, distinct from peptide fragment peaks. The pattern of peaks in the final mass spectrum is matched to a database in order to identify the parent peptide. The intensities of the reporter ion peaks indicate the relative abundance of the four samples.
Article Snippet: Each peptide mixture was separated on an SCX chromatography column (Polysulfoethyl column, The Nest Group Inc, Southborough, MA USA).
Techniques: Sequencing, Blocking Assay, Labeling, Multiplex sample analysis, Fractionation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy
