Targeting deeper the human serum fucome by a liquid-phase multicolumn platform in combination with combinatorial peptide ligand libraries

Abstract

Combinatorial peptide ligand library (CPLL) was evaluated as an off line step to narrow the differences of protein concentration in human serum prior to the capturing of human fucome from disease-free and breast cancer sera by a multicolumn platform via lectin affinity chromatography (LAC) followed by the fractionation of the captured glycoproteins by reversed phase chromatography (RPC). Two monolithic lectin columns specific to fucose, namely Aleuria aurantia lectin (AAL) and Lotus tetragonolobus agglutinin (LTA) columns were utilized to capture the fucome, which was subsequently fractionated by RPC yielding desalted fractions in volatile acetonitrile-rich mobile phase, which after vacuum evaporation were subjected to tryptic digestion prior to LC-MS/MS analysis. AAL has a strong affinity towards core fucosylated N-glycans and has a weak binding towards fucose in the outer arm while LTA can bind to glycans having fucose present in the outer arm. The combined strategy consisting of the CPLL, multicolumn platform and LC-MS/MS analysis permitted the identification of the differentially expressed proteins (DEPs) in breast cancer serum yielding 58 DEPs in both the LTA and AAL fractions with 6 DEPs common to both lectins. 17 DEPs were of the low abundance type, 16 DEPs of the borderline abundance type, 4 DEPs of the medium abundance type and 15 DEPs of the high abundance type. The remaining 6 DEPs are of unknown concentration. Only proteins exhibiting 99.9% protein identification probability, 95% peptide identification probability, and a minimum of 5 unique peptides were considered in finding the DEPs via scatterplots.

Capturing and identification of differentially expressed fucome by a gel free and label free approach

“…In this work, which is a continuation to our recent investigations [26, 27], we intend to feature three new elements of the recently developed multicolumn platform: (i) the effectiveness of the platform in mapping the altered fucome in another cancer serum, namely human serum with HCC, (ii) reveal clearly the instrumental aspect of the platform for comparative proteomics that does not involve labeling chemistry and consequently requires much less labor than existing technology, and (iii) demonstrate the superiority of the described method in generating the whole fucome as compared to partial fucome by the combination of other methodologies. Regarding aim (i), since every cancer develops and behaves differently, it is therefore important to challenge the developed strategy in revealing similarities and differences in the altered fucome in HCC with respect to that in breast cancer.…”

“…In fact, when compared to the previous work from our laboratory [27] that involved the capturing of human fucome from disease-free and breast cancer sera using similar multicolumn platform in combination with the off line protein equalization via the CPLL approach, a total of 70 DEPs in HCC serum were found in the current study versus 58 DEPs in breast cancer serum were found in the previous study. There were 35 and 23 unique DEPs to the current and previous study, respectively, see Table S7 in Supporting information.…”

“…The DEPs were considered from the proteins that were more than two standard deviations away from being the same in both categories, and all these proteins had a p-value < 0.05 using the t-test. As reported earlier by Selvaraju and El Rassi, the Q-Q scatterplots proved very reliable in the identification of DEPs [26, 27]. Some selected Q-Q scatterplots for the RPC fractions originating from both LTA and AAL lectin columns for equalized and untreated serum are provided in Supporting information (See Figs.…”

“…1b and 2b). For both equalized and untreated sera, the DEPs in the HCC serum relative to the disease-free serum were considered if they could be established for at least 99.9% protein identification probability, 95% peptide identification probability, and contained at least 5 unique peptides [26, 27]. The DEPs were identified using the Q-Q scatterplot which plots the normalized spectral count for each protein found in the HCC serum versus the normalized spectral count of the same protein found in disease-free serum.…”

“…Five mL of each serum samples (disease-free or cancer serum) were treated by the CPLL beads according the manufacturer instructions, and the procedure was also described very recently by Selvaraju and El Rassi [27]. The treated (i.e., equalized) disease-free or HCC serum was stored at −20 ºC until use.…”