Chip‐LC‐MS for label‐free profiling of human serum

Abstract

The discovery of biomarkers in easily accessible body fluids such as serum is one of the most challenging topics in proteomics requiring highly efficient separation and detection methodologies. Here, we present the application of a microfluidics-based LC-MS system (chip-LC-MS) to the label-free profiling of immunodepleted, trypsin-digested serum in comparison to conventional capillary LC-MS (cap-LC-MS). Both systems proved to have a repeatability of approximately 20% RSD for peak area, all sample preparation steps included, while repeatability of the LC-MS part by itself was less than 10% RSD for the chip-LC-MS system. Importantly, the chip-LC-MS system had a two times higher resolution in the LC dimension and resulted in a lower average charge state of the tryptic peptide ions generated in the ESI interface when compared to cap-LC-MS while requiring approximately 30 times less (~5 pmol) sample. In order to characterize both systems for their capability to find discriminating peptides in trypsin-digested serum samples, five out of ten individually prepared, identical sera were spiked with horse heart cytochrome c. A comprehensive data processing methodology was applied including 2-D smoothing, resolution reduction, peak picking, time alignment, and matching of the individual peak lists to create an aligned peak matrix amenable for statistical analysis. Statistical analysis by supervised classification and variable selection showed that both LC-MS systems could discriminate the two sample groups. However, the chip-LC-MS system allowed to assign 55% of the overall signal to selected peaks against 32% for the cap-LC-MS system.

Microfluidic sensing: state of the art fabrication and detection techniques

“…[103][104][105][106][107][108][109][110][111][112][113] Among these methods, optical and electrochemical methods are the most frequently utilized due to their selectivity and sensitivity. Other than the above major methods, approaches such as nuclear magnetic resonance (NMR) spectroscopy, [114][115][116][117][118] magnetoresistive, 119 and acoustical 120,121 are also coupled to microfluidics for sensing application.…”

“…To date, several MS configurations have already been developed to be integrated with microfluidic devices for this application, for instance, electrospray ionization (ESI)-MS (Refs. 104-106) and matrix-assisted laser desorption ionization (MALDI)-MS. [107][108][109] In 2006, another paradigm is ion trap mass spectrometry integrated with microfluidics for protein identification by Hardouin et al 111 Other systems were also reported, such as a chip-liquid chromatography (LC)-MS system for labelfree profiling of human serum 113 and a LC-ESI-MS system for multiple proteins detection from breast cancer cellular extract. 112 The resolving ability of MS has recently been pushed down to the detection of a single molecular by Roukes's group in Caltech using a nanoelectromechanical system-based MS (NEMS-MS) (Fig.…”

Environmental Proteomics: a Paradigm Shift in Characterizing Microbial Activities at the Molecular Level

“…The overall performance of the microfluidic chip enabled a gradient liquid chromatographic separation and MS/MS identification of peptides with an estimated detection sensitivity of 5 to 7 fmol, demonstrated with both a plant and the human tissue sample (50). Horvatovich et al compared label-free profiling of immunodepleted, trypsin-digested serum by a microfluidics-based LC-MS system with a conventional capillary LC-MS system (18). The chip-LC-MS system had a two-times-higher resolution on the LC dimension and resulted in a lower average charge state of the tryptic peptide ions generated in the ESI interface than the cap-LC-MS while requiring about 30-times-less sample (18).…”

Use of DNA adducts to identify human health risk from exposure to hazardous environmental pollutants: The increasing role of mass spectrometry in assessing biologically effective doses of genotoxic carcinogens

“…These use microfluidics-based technology for high-pressure nanoflow LC-MS systems, and incorporate the sample purification, separation and electrospray interface on a single chip thus allowing the processing of lower volume samples with complete automation of the analysis procedure and the possibility of screening based applications [80,81,82].…”