Automated analysis of mouse serum peptidome using restricted access media and nanoliquid chromatography-tandem mass spectrometry.

We demonstrate use of restricted access media with reversed phase functionality (RAM-RP) for analysis of low molecular weight proteins and peptides in mouse serum (75 µl) using a custom designed modular automated processing system (MAPS). RAM-RP fractionation with simultaneous removal of high molecular weight and high abundance proteins is integrated with a follow-on buffer exchange module (BE) to ensure compatibility with subsequent processing steps (trypsin digestion and intact peptide separation prior to mass spectrometric analysis). The high sample capacity afforded by chromatographic methods generates enough sample to achieve comprehensive serum peptidome identification (357 proteins) through tandem mass spectrometric analysis of both intact and digested peptides. Sample losses during transfer between modules are minimized through precise fluidic control; no clogging occurred over several months of serum processing in our low back pressure system. Computer controlled operation of both modules and thorough optimization yield excellent run-to-run reproducibility and protein/peptide overlap in analytical repeats. The robustness of our results demonstrate that the RAM-RP-BE workflow executed on our MAPS platform shows tremendous potential for high throughput peptidome processing, particularly with regard to direct analysis of small-volume serum samples.

Nipple-areolar pigmentation: histology and potential for reconstitution in breast reconstruction.

The makeup of nipple-areolar skin, in terms of its melanin and melanocyte content has not previously been established. This histological information is required if pigmentation of the reconstructed nipple-areola is to be successful in post-mastectomy breast reconstruction. We describe examination of 200 parallel sections of nipple-areolar skin of 20 women using histochemical (Masson-Fontana) and immunohistochemical (Mel-5) techniques, evaluated using quantitative image analysis. The amount of melanin present per length of basement membrane was 2.14 times higher in areolar skin than breast skin. The ratio of melanocytes to keratinocytes was 1:9.7 in areolar skin vs. 1:14.7 in breast skin. We also describe a cell culture and skin construct method using autologous human serum without toxic growth promoting additives, which could be used in the clinical setting of nipple-areolar reconstruction.