Screening the low molecular weight fraction of human serum using ATR-IR spectroscopy

Abstract

Vibrational spectroscopic techniques can detect small variations in molecular content, linked with disease, showing promise for screening and early diagnosis. Biological fluids, particularly blood serum, are potentially valuable for diagnosis purposes. The so-called Low Molecular Weight Fraction (LMWF) contains the associated peptidome and metabolome and has been identified as potentially the most relevant molecular population for disease-associated biomarker research. Although vibrational spectroscopy can deliver a specific chemical fingerprint of the samples, the High Molecular Weight Fraction (HMWF), composed of the most abundant serum proteins, strongly dominates the response and ultimately makes the detection of minor spectral variations a challenging task. Spectroscopic detection of potential serum biomarkers present at relatively low concentrations can be improved using pre-analytical depletion of the HMWF. In the present study, human serum fractionation by centrifugal filtration was used prior to analysis by Attenuated Total Reflection infrared spectroscopy. Using a model sample based on glycine spiked serum, it is demonstrated that the screening of the LMWF can be applied to quantify blinded concentrations up to 50 times lower. Moreover, the approach is easily transferable to different bodily fluids which would support the development of more efficient and suitable clinical protocols exploring vibrational spectroscopy based ex-vivo diagnostic tools

Ultra-filtration of human serum for improved quantitative analysis of low molecular weight biomarkers using ATR-IR spectroscopy

“…Figure 2 presents the dependence of the area under the curve observed in raw data (AUC -between baseline and peak maximum), calculated for the band 1180-955 cm -1 , depending on deposited volume, for two different concentrations of glucose solution. The 10 g.dL -1 solution does not exhibit any noticeable decrease in the AUC between 0.5 µL and 2µL, explained by a full coverage of the crystal coupled to a saturation of the signal 24 . The notion of saturation is defined by the loss of linearity between the absorbance and the concentrations measured.…”

“…In the present study, a 10 kDa centrifugal device was used in order to specifically deplete the abundant proteins such as globulins and albumin 24 and retain only the molecular species with low molecular weight in the filtrate, below the cut off point of the membrane, for subsequent spectroscopic analysis. For instance, glucose is about 180 Daltons, which means it will freely pass through the membrane during spinning of the human serum.…”

“…Following air drying, the human serum spectrum displays numerous well defined peaks within the 4000-650 cm -1 spectral widow, allowing clear identification of specific features of the human serum, for example those highlighted in grey at 3280 cm -1 (H-O-H stretching), 2957 cm [53][54][55] . The spectrum from whole human serum is clearly dominated by the abundant proteins contribution such as albumin and globulins 24 which swamp the contribution of less represented biomolecules. In comparison, the glucose signature exhbits fewer features, the main peaks being located in the spectral range 1280 -800 cm -1 , resulting from both the ν(C-O) and ν(C-O-C) vibrational modes 56 , as identified in Figure 1A.…”

“…The advent of imaging technologies coupled to the rapid data collection offered by FTIR systems has contributed considerably to the attraction of infrared absorption spectroscopy for biomedical applications [21][22][23] . However, other methods such as ATR-FTIR are highly suitable for analysis of liquid samples, such as body fluids, allowing delivery of chemical fingerprints from micro-deposition of the samples directly on the Attenuated Total Reflection (ATR) crystal 24,25 . The IR spectrum contains rich and specific information about the molecular composition of the human serum which, coupled to advanced multivariate analysis tools, could deliver accurate diagnostics.…”

“…Therefore, depletion of the highly abundant proteins is now recognized as the first step, yet to be optimized, in the analysis of the serum composition 42 . ATR-FTIR suffers from similar limitations to those encountered with chromatography and/or mass spectroscopy based approaches, which can be overcome by means of sample fractionation prior to data recording 24 . Including a separation step prior to IR analysis in order to enhance the specificity and sensitivity have been proposed trough coupling with chromatography technologies such as LC-IR and GC-IR 43 .…”