Optimizing the SWATH-MS-workflow for label-free proteomics.

Quantification of proteins by mass spectrometry at the level of entire proteomes has become a central aspect in contemporary proteomics. Sequential Window Acquisition of all THeoretical fragment-ion spectra (SWATH) mass spectrometry is a label-free protein quantification method enabling parallel quantification of up to several thousand proteins, thereby granting a comprehensive overview of highly complex samples. We optimized several parameters of the mass spectrometrical detection to increase the size of the library, enhance the precision of protein quantifications and decrease systematic errors induced by fragment ion interference. By these means enhanced differential analyses facilitate the detection of more significantly regulated proteins with a dynamic range of over three orders of magnitude.

A FRET based pH probe with a broad working range applicable to referenced ratiometric dual wavelength and luminescence lifetime read out.

A luminescent probe for determination of pH was designed based on a Förster resonance energy transfer (FRET) system, combining a europium chelate as the donor and carboxynaphtho-fluorescein as a pH sensitive acceptor. The FRET system enables referenced pH detection in an exceptional broad dynamic range from pH 3 to 9.

Background-free referenced luminescence sensing and imaging of pH using upconverting phosphors and color camera read-out.

Fluorescence background and problems with proper signal referencing severely disrupt the read-out of luminescence sensors and images. We present a pH sensor film in combination with a simple read-out technique that overcomes issues of background signals and autofluorescence. It consists of micrometer-sized upconversion phosphors (UCPs) and a pH indicator (Neutral Red) that absorbs their green emission. Both are embedded in a proton permeable hydrogel matrix. The UCPs generate green and red luminescence upon excitation with IR light of 980 nm wavelength. Solely the green light of the UCPs is affected by the pH indicator, while the red emission acts as inert reference signal for ratiometric measurements. The emission peaks of the UCPs match the red and green color channels of standard digital cameras. Thereby, the devised sensor film can be used for referenced ratiometric sensing and 2D imaging of pH using a color camera read-out. The sensor setup using common and hand-held devices is cheap and straightforward and allows for point-of-care measurements. Finally, pH measurements in human serum samples show the potential of this sensor for imaging free of interfering background signals.