Evaluation of Endogenous Plasma Peptide Extraction Methods for Mass Spectrometric Biomarker Discovery

Abstract

Peptides have a role in the inflammatory response, tumor biology, and endocrine processes, presenting them as appealing biomarker candidates. However, peptide extraction efficacy for clinical profiling remains a pivotal technological challenge, as maximum coverage of the plasma peptidome is limited by a range of factors including the inherent complexity of human plasma and the lower concentration of peptides compared to abundant proteins. The aim of this study was to evaluate commonly employed peptide extraction methodologies in terms of total number of peptides detected and the mass range of peptides observed by MALDI. Despite showing coelution of proteins, solid-phase extraction (SPE) methods exhibited superior plasma peptide recovery than ultrafiltration, acetonitrile (ACN) precipitation, or size-exclusion chromatography methods under conditions employed in the study. Not surprisingly, in line with studies challenging the veracity of many peptide biomarker studies, the majority of identified peptides eluted from SPE methods corresponded to proteolytic truncations of the most abundant plasma proteins. The prefractionation of plasma with acetonitrile precipitation prior to SPE provided distinct ion signal profiles and is worthy of further study. In conclusion, this study favors the use of SPE in peptide extraction protocols for increased biomarker coverage and diversity from the plasma peptidome.

Mass spectrometry of peptides and proteins from human blood

“…At present hundreds to thousands of endogenous peptides, including peptides from low abundance blood proteins, can be routinely extracted and identified from blood fluids (Tucholska et al, 2007;Williams et al, 2007;Hu et al, 2009a). There has been an intense effort to identify (Marshall et al, 2003 and examine the low molecular mass polypeptides of blood, sometimes referred to as the peptidome (Verhaert et al, 2001;Jurgens & Schrader, 2002;Tirumalai et al, 2003;Merrell et al, 2004;Villanueva et al, 2004Villanueva et al, , 2006aVillanueva et al, ,b, 2007Villanueva et al, , 2008Ping et al, 2005;Rai et al, 2005;Tammen et al, 2005aTammen et al, ,b, 2007Falth et al, 2006Falth et al, , 2008Geho et al, 2006;Govorukhina et al, 2006;Hortin, 2006;Ogata et al, 2006;Huang et al, 2006b;Zheng, Baker, & Hancock, 2006b;Aristoteli, Molloy, & Baker, 2007;Tian et al, 2007;Tomonaga & Nomura, 2007;Tucholska et al, 2007;Hernandez et al, 2008;Kay et al, 2008;Kojima et al, 2008;Ly & Wasinger, 2008;Mayer, Poirier, & Seidah, 2008;Navare et al, 2008;Pernemalm et al, 2008;Plavina et al, 2008;Ward et al, 2008;Xia, Wu, & Jemal, 200...…”

“…A simple and effective form of liquid partition chromatography is the cast membrane structure invented by William Kopaciewicz of Millipore Corporation commonly called the C18 ziptip (Aristoteli, Molloy, & Baker, 2007;Jin et al, 1999). Microchromatography may be used directly prior to MS/MS analysis of blood fluids by MALDI or ESI (Marshall et al, 2003;Tiss et al, 2007;Navare et al, 2008).…”

Feasibility of electrodialysis as a fast and selective sample preparation method for the profiling of low‐abundant peptides in biofluids

“…Actually, the ED device and method were not fully optimized to achieve maximal sensitivity yet; more peptides are expected to be detected when further improvements as discussed throughout the paper will be implemented. As discussed by Aristoteli, each sample preparation method has a bias toward certain peptide characteristics, producing its own unique set of peptide ion masses [6]. The choice of method should therefore be guided by the properties of the peptides of interest.…”

“…In practice, the sample preparation techniques that are most frequently used for peptide profiling studies are ultrafiltration, SPE, and precipitation. Aristoteli et al compared these preparation techniques for plasma with respect to the number of peptides detected and the Jurre J. Kamphorst 1,2 Ubbo R. Tjaden 1 mass range of peptides observed by MALDI-TOF-MS [6]. From this work it became clear that each preparation technique produces its own unique set of peptide ion masses, indicating that none of the techniques can profile all the peptides present in the sample, and that each technique has certain limitations.…”

“…For example, SPE not only generates the highest recoveries but also a lot of protein contaminations are observed. Ultrafiltration and precipitation provide cleaner extracts, but the recovery of the peptides is generally lower [6]. In addition, both methods tend to be slow and are less suitable for automation and/or high-throughput applications.…”