Compared with immunodepletion alone, our integrated strategy substantially improved the proteome coverage afforded by PEG fractionation

Compared with immunodepletion alone, our integrated strategy substantially improved the proteome coverage afforded by PEG fractionation. (XLSX) pone.0166306.s003.xlsx (146K) GUID:?69549602-11F9-43F2-ACC9-0CDDB5D6D7B2 S2 Table: 135 known LAPs with reported concentrations below 100 ng/mL (based on previously published data) identified by LC-MS/MS in this study. (XLSX) pone.0166306.s004.xlsx (17K) GUID:?9D46D71F-8AD6-4832-986D-D5A3B356902F Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract The enormous depth complexity of the human plasma proteome poses a significant challenge for current mass spectrometry-based proteomic technologies in terms of detecting low-level proteins in Atopaxar hydrobromide plasma, which is essential for successful biomarker discovery efforts. Typically, a single-step analytical approach cannot reduce this intrinsic complexity. Current simplex immunodepletion techniques offer limited capacity for detecting low-abundance proteins, and integrated strategies are thus desirable. In this respect, we developed an improved strategy for analyzing the human plasma proteome by integrating polyethylene glycol (PEG) fractionation with immunoaffinity depletion. PEG fractionation of plasma proteins is simple, rapid, efficient, and compatible with a downstream immunodepletion step. Compared with immunodepletion alone, our integrated strategy substantially improved the proteome coverage afforded by PEG fractionation. Coupling this new protocol with liquid chromatography-tandem mass spectrometry, 135 proteins with reported normal concentrations below 100 ng/mL were confidently identified as common low-abundance proteins. A side-by-side comparison indicated that our integrated strategy was increased by average 43.0% in the identification rate of low-abundance proteins, relying on an average 65.8% increase of the corresponding unique peptides. Further investigation demonstrated that this combined strategy could effectively alleviate the signal-suppressive effects of the major high-abundance proteins by affinity depletion, especially with moderate-abundance proteins after incorporating PEG fractionation, thereby greatly enhancing the detection of low-abundance proteins. In sum, the newly developed strategy of incorporating PEG fractionation to immunodepletion methods can potentially aid in the discovery of plasma biomarkers of therapeutic and clinical interest. Introduction Recently, the discovery of reliable disease biomarkers is one of the biggest concerns for researchers [1C5]. Human blood plasma Rabbit Polyclonal to hnRNP L is one of the most studied biological fluids and is the main sample type used for disease diagnosis [6C8]. In contrast to brain tissue and cerebrospinal fluid, human plasma can be sampled easily without invasive procedures. Importantly, it contains numerous proteins that are actively secreted or offered following cell and cells leakage [3]. These proteins are involved in protein transport, immune defense mechanisms, coagulation, and protease inhibition, and their levels can provide an indication of an individuals physiological or pathological claims [9C11]. Mass spectrometry (MS)-centered plasma proteomics is definitely a useful means for identifying novel medical biomarkers that can provide unambiguous protein assignments [12]. However, because of the huge difficulty and different concentrations of component proteins, the analysis of plasma proteome faces the great difficulties [3]. Moreover, the detection limits of MS imposed by the various ionization processes used impact both the complexity and dynamic range of analytes that are measurable [13]. These disadvantages are reflected from the predominance of high- and moderate-abundance proteins (HAPs and MAPs), which clearly hampers the recognition and quantification of potential low-abundance protein (LAP) biomarkers [2]. As a result, the LAPs among a large excess of Atopaxar hydrobromide additional proteins, recognized by MS is definitely key for studying particular diseases both with respect to the limited amount of protein analyzed and to the given quantity of particular proteins in the sample [14]. During the last few decades, the most commonly used approach to facilitate LAP analysis has been to reduce plasma sample complexities by fractionation [15]. Considerable effort has Atopaxar hydrobromide been directed at improving fractionation strategies with human being plasma samples, and many different techniques have been developed and applied [16]. A current popular approach is definitely immunoaffinity depletion (IAD), in which antibodies are used to capture probably the most abundant proteins [17]. Immunodepletion can facilitate the analysis of the next tier of proteins by eliminating some of the most abundant proteins. This effective strategy is definitely progressively becoming applied in various biomarker studies [1, 18]. Alternatively, protein ultrafiltration and precipitation strategies, discriminated by size or size/pI, respectively, have also been widely applied prior to sample profiling by electrophoresis or MS [19, 20]. Centrifugal ultrafiltration.