Deep proteome profiling of sera from never-smoked lung cancer patients

Abstract

Previous studies on the serum proteome are hampered by the huge dynamic range of concentration of different protein species. The use of Equalizer Beads coupled with a combinatorial library of ligands has been shown to allow access to many low-abundance proteins or polypeptides undetectable by classical analytical methods.

This study focused on never-smoked lung cancer, which is considered to be more homogeneous and distinct from smoking-related cases both clinically and biologically. Serum samples obtained from 42 never-smoked lung cancer patients (28 patients with active untreated disease and 14 patients with tumor resected) were compared with those from 30 normal control subjects using the pioneering Equalizer Beads technology followed by subsequent analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS).

Eighty-five biomarkers were significantly different between lung cancer and normal control. The application of classification algorithms based on significant biomarkers achieved good accuracy of 91.7%, 80% and 87.5% in class-prediction with respect to presence or absence of disease, subsequent development of metastasis and length of survival (longer or shorter than median) respectively. Support vector machine (SVM) performed best overall.

We have proved the feasibility and convenience of using the Equalizer Beads technology to study the deep proteome of the sera of lung cancer patients in a rapid and high-throughput fashion, and which enables detection of low abundance polypeptides/proteins biomarkers. Coupling with classification algorithms, the technologies will be clinically useful for diagnosis and prediction of prognosis in lung cancer.

Introduction

Most phenotypic manifestations are believed to be related to protein expressions, which are not predictable from the knowledge of the genome alone. The ‘proteome’ reflects the state of a cell, tissue or organism more accurately and therefore from it we are more likely to discover better tumour markers for disease diagnosis and therapy monitoring. The application of mass spectrometry (MS) in proteomic research is a major technological breakthrough and enables high-throughput analysis of protein mixtures.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was especially designed for biomarker discovery and has become a popular tool for protein pattern profiling. The concept of surface-enhanced laser desorption/ionization (SELDI) was introduced by Hutchens and Yip [1] in 1993 and this novel strategy of mass spectrometric analysis of macromolecules simplified sample extraction and facilitated effective on-probe investigation of biopolymers when compared to conventional laser desorption/ionization (e.g. MALDI). Since the presentation of this landmark work, many exciting SELDI-TOF-MS applications have been described [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20].

Serum is more easily available than tissue and hence holds the promise of a revolution in early disease detection, predicting biological aggressiveness and prognosis, and therapeutic monitoring. However, serum is a complex biological mixture comprising hundreds of thousands of different polypeptides of an estimated dynamic range of over ten orders of magnitude (Fig. 1). In addition to the “classical serum proteins” (albumin, immunoglobulins, transferrin, etc.), it contains all tissue proteins (as leakage markers) plus cytokines [21]. Therefore, high-throughput proteomic analyses are greatly hampered by the huge dynamic range of concentration of different protein species, making the detection of trace proteins difficult [22].

The novel use of Equalizer Beads coupled with a combinatorial library of ligands [23] have been shown to allow access to many low-abundance proteins or polypeptides undetectable by classical analytical methods [24], [25]. The population of beads has such diversity that a binding partner should exist for most proteins in a sample. Each bead has an equivalent binding capacity. High abundance proteins saturate their binding partner and excess protein is washed away, whereas trace proteins are concentrated on their specific ligands (Fig. 2). Coupled with SELDI-TOF-MS, it is a new approach for the rapid and simultaneous resolution of multiple low-abundance proteins.

For lung cancer, various attempts [4], [12], [26], [27] have been made to profile the proteomes using MS. The present study is the first to use the Equalizer Beads technology to study the proteome especially the low-abundance proteins in the sera of never-smoked lung cancer patients, which is considered as a more homogeneous and distinct disease group amongst different etiological types of lung cancer [28]. As the serum proteins of lung cancer patients may be either due to the presence of the tumour itself or the host response, it may also be enlightening to compare the sera of those patients with freshly diagnosed untreated lung cancer and those patients who had the tumour already completely resected.