Mass spectrometrical analysis of the processed metastasis-inducing anterior gradient protein 2 homolog reveals 100% sequence coverage.

Anterior gradient protein 2 homolog is a metastasis-inducing protein in a rat model of rat breast cancer and prognostic for outcome in hormonally treated breast cancer patients. Carrying out protein profiling in several mammalian cells and tissues, we detected this protein (synonym: secreted cement gland protein XAG-2 homolog) that was originally described in toad skin, in human bronchial epithelia. Tissues obtained from biopsies were homogenised and extracted proteins were run on two-dimensional gel electrophoresis. Following in-gel digestion with proteases trypsin, AspN, LysC and chymotrypsin, mass spectrometrical analysis was carried out by MALDI-TOF/TOF. The use of MS following multi-enzyme digestion of the protein resulted into 100% sequence coverage. MS/MS analysis enabled sequencing of 87% of the protein structure. This percentage does not include the signal peptide that was not observed in our protein due to processing. No posttranslational modifications were detectable and no sequence conflicts were observed. Complete analysis, unambiguous identification and characterisation of this biologically important protein could be shown, which is relevant for the definition of a marker protein that has been described so far by immunochemical methods only. Complete analysis is of importance as it forms the basis for all future work on this protein and, moreover, may serve as an analytical tool for further studies.

Deranged hypothetical proteins Rik protein, Nit protein 2 and mitochondrial inner membrane protein, Mitofilin, in fetal Down syndrome brain.

Down syndrome (DS) is the most common genetic disorder with mental retardation and a host of deranged proteins has already been described. Protein hunting leads to rapid accumulation of aberrant proteins and proteomics methods not only allow unambiguous identification of proteins, they are also a powerful tools to identify new or predicted proteins. We applied two-dimensional gel electrophoresis with in-gel digestion of proteins and subsequent MALDI-TOF mass-spectrometrical identification and quantification of spots using specific software on cortical brain samples from 7 controls and 7 samples from fetal DS at the early second trimester. Nine hypothetical proteins were identified: three of them (4833418L03Rik protein Q9D614, mitochondrial inner membrane protein Q16891 and Nit protein 2 Q8WUF0) were significantly and about doublefold reduced in fetal DS brain. Hypothetical proteins CGI 99, FLJ10463, 70 kDa WD-repeat tumor rejection antigen homolog, KSRP, Hypothetical protein 49.6 kDa and Elongin A were comparable between groups. Domain analysis of deranged structures revealed a t_SNARE domain for the Rik protein, indicating involvement of this protein in the exocytotic-synaptic machinery impaired in DS, a CN hydrolase domain for Nit protein 2, possibly reflecting aberrant nitrilase-related metabolism and handling and an inner mitochondrial protein, extending knowledge on the mitochondrial deficit in in fetal DS early in life.