Journal of Chromatography A, 1232 (2012) 276–280 Contents lists available at SciVerse ScienceDirect Journal of Chromatography A j our na l ho me p ag e: www.elsevier.com/locate/chroma The utility of porous graphitic carbon as a stationary phase in proteomics workflows: Two-dimensional chromatography of complex peptide samples John R. Griffiths a,∗,1 , Simon Perkins a,1 , Yvonne Connolly a , Lu Zhang a , Mark Holland a , Valeria Barattini b , Luisa Pereira b , Anthony Edge b , Harald Ritchie b , Duncan L. Smith a a Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, UK b Thermo Fisher Scientific, Tudor Road, Manor Park, Runcorn, Cheshire, UK a r t i c l e i n f o Article history: Available online 13 January 2012 Keywords: Peptide fractionation Porous graphitic carbon Strong cation exchange Proteomics, 2D-LC–MS/MS a b s t r a c t We present the first investigation into the utility of porous graphitic carbon (PGC) as a stationary phase in proteomic workflows involving complex samples. PGC offers chemical and physical robustness and is capable of withstanding extremes of pH and higher temperatures than traditional stationary phases, without the likelihood of catastrophic failure. In addition, unlike separations driven by ion exchange mechanisms, there is no requirement for high levels of non-volatile salts such as potassium chloride in the elution buffers, which must be removed prior to LC–MS analysis. Here we present data which demonstrate that PGC affords excellent peptide separation in a complex whole cell lysate digest sample, with good orthogonality to a typical low pH reversed-phase system. As strong cation exchange (SCX) is currently the most popular first dimension for 2D peptide separations, we chose to compare the perfor- mance of a PGC and SCX separation as the first dimension in a comprehensive 2D-LC–MS/MS workflow. A significant increase, in the region of 40%, in peptide identifications is reported with off-line PGC fraction- ation compared to SCX. Around 14,000 unique peptides were identified at an estimated false discovery rate of 1% (n = 3 replicates) from starting material constituting only 100 g of protein extract. © 2012 Elsevier B.V. All rights reserved. 1. Introduction The comprehensive analysis of complex peptide mixtures, such as those derived from a whole cell lysate (WCL), by LC–MS/MS represents a significant challenge due to acquisition rate limita- tions and consequent under sampling by current generation mass spectrometers. Since the mass spectrometer only has a finite time (<30 s elution time) in which to detect (MS/MS) co-eluting peptides, many peptides which co-elute result in some of them remain- ing undetected within that time window. Pre-fractionation prior to RPLC–MS/MS such as in a MudPit approach is often used as a way of increasing the number of peptide (and associated protein) identifications [1]. Strong cation exchange (SCX) chromatography is currently the most widely utilized first dimension for such analy- ses [2–4]. However, SCX requires the use of high salt concentrations which need extensive removal prior to electrospray ionization. It has also been shown that peptides tend to group in relatively few SCX fractions due to the low resolving power of the intrinsic ∗ Corresponding author. Tel.: +44 01614463155; fax: +44 01614463109. E-mail address: jgriffiths@picr.man.ac.uk (J.R. Griffiths). 1 These two authors contributed equally to this work. mechanism, which separates according to their solution-phase charge [5]. As an alternative to SCX, separation on a reversed-phase col- umn at elevated pH (in the region of pH 10.5) has been used [6–8]. This mode of separation offers higher resolution than SCX, and has proved very effective at enabling deeper proteome penetrance compared to those previously observed [9]. Since most reversed- phase columns are based upon a silica support, the challenge to manufacturers is to produce columns capable of withstanding alkali conditions (pH 10.5) which would normally dissolve silica. In addition, the chromatographic system itself must be capable of withstanding these higher pH conditions. The column and system compatibility issues at elevated pH have limited the utility of this approach in the proteomics community. Recently, McNulty and Annan described the use of an alterna- tive separation mode for the first stage of global enrichment of phosphopeptides, termed hydrophilic interaction chromatography (HILIC) [10] first described by Alpert [11]. Gilar et al. reported the potential of HILIC as a suitable chromatography separation mode with good orthogonality to reversed-phase [5]. However, since solutes are required to be dissolved in high organic solvents (70% acetonitrile) the solubility of certain peptides in such systems may be problematic. SCX is therefore still the dominant first dimension of choice in these 2D peptide separations despite its limitations. 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2012.01.015