Development of a Univariate Membrane-Based Mid-Infrared Method for Protein Quantitation and Total Lipid Content Analysis of Biological Samples

Figure 5

Application of the MIR-based univariate method for sample optimization in a short study of breast cancer biomarkers. The method was used to monitor the efficiency of fat removal and total protein liberation at each step of sample preparation from breast cancer tissue. Briefly, equal portions of breast cancer tissue were initially homogenized using either of two lysis buffers (RIPA or CytoBuster). Following homogenization, the samples were subjected to successive centrifugation steps. Resulting fractions were analyzed for protein and lipid content using the MIR-based method. (a) Overlay of raw MIR spectra collected for fraction 1 (P1a, L1b, and P1b, see below for definition) of breast cancer tissue lysed using RIPA buffer (violet, red, and brown) and CytoBuster protein extraction reagent (blue, cyan, and navy). In order to improve visualization, sets of curves representing each lysis buffer were manually separated. Areas utilized to quantify protein and analyze lipids are highlighted. (b) Total protein recovery across the centrifugal fractions. P1a and P2a represent bottom layer fraction from the first centrifugal spin. L1b and L2b represent top lipid containing fraction from the second spin. P1b and P2b show protein content in bottom layer fraction from second centrifugal spin. Total protein liberated using CytoBuster protein extraction reagent is shown in black while protein recovered using RIPA buffer is shown in grey. (c) Efficiency of gradual fat removal by consecutive spinning cycles. Fractions shown are the same as presented in B. Relative absorbance of lipid using CytoBuster protein extraction reagent is shown in black while protein recovered using RIPA buffer is shown in grey. Dual analysis of protein yield and fat content permits in-line optimization of the sample preparation process to meet the requirements of each downstream method of analysis.

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