Pancreatic cancer is the fourth most common cause of cancer-related mortality in the United States.(1
) The five-year survival rate is the lowest among all cancers, with estimates ranging from 0.4 to 4%. In 2007, an estimated 37
170 new cases of pancreatic were diagnosed, and an estimated 33
370 patients died as a result of their disease.(2
) No effective screening biomarker exists for the early detection of pancreatic cancer. Carbohydrate antigen (CA) 19-9 is the most commonly used protein tumor marker for pancreatic cancer, however its use is largely limited to following the course of disease.3,4
This is primarily because CA 19-9 can be expressed in benign conditions such as cholangitis and chronic pancreatitis.5,6
Furthermore, CA 19-9 is not expressed at all by some pancreatic tumors, and in other tumors, it is often not detectable until pancreatic cancer is at a late and incurable stage.(7
) Other potential biomarkers such as carcinoembryonic antigen (CEA), peanut agglutinin (PNA)-binding glycoproteins,(8
(telomerase catalytic subunit),(9
) and matrix metalloproteinase-2 (MMP-2)(10
) also lack clinical efficacy. This situation has stimulated our search for biomarkers that can be used for the early detection of pancreatic cancer.(11
Despite significant advances in proteomic methods and instrumentation, discovery of circulating disease biomarkers remains extremely challenging. We have developed a novel approach for identifying biomarkers of pancreatic cancer in human serum. The approach specifically addresses three of the major obstacles in biomarker discovery.
The first major obstacle is the accurate quantitation of large numbers of proteins. The difficulty of accurate protein quantitation is compounded by nonspecific losses suffered during extensive sample processing. For example, although immunoaffinity purification removes the major abundant serum proteins, significant losses of low abundant proteins bound to the high abundant proteins can occur.(12
) Therefore, a stable isotope labeled proteome (SILAP) standard added to serum samples prior to immunopurification can act as a carrier for the low abundance proteins, controlling for and minimizing the possibility of such losses.13,14
The SILAP standard can also control for and help to prevent losses that can occur throughout the extensive workup procedure and LC-MS analysis, such as the nonspecific binding of peptides to glassware and surfaces.(15
) Equally important, for any protein identified in the SILAP standard, the corresponding unlabeled serum protein can be quantified, if present. Absence of the unlabeled serum protein when the labeled protein is identified confers increased confidence that the protein is truly absent in the serum sample, as opposed to simply absent from the analysis due to sampling error, ion suppression or nonspecific loss.
The second major obstacle is the identification and characterization of biologically relevant proteins in serum. In unbiased shotgun analyses of serum samples, many of the proteins found to be differentially expressed are abundant serum proteins or nonspecific acute phase proteins, and not proteins related to the disease process. By using a SILAP standard derived from the secreted proteome of the CAPAN-2 pancreatic cancer cell line, low abundance and biologically relevant biomarker candidates can be identified and relatively quantitated. These proteins are generally present at much lower concentrations in serum than in the secreted CAPAN-2 proteome, making their quantitation and identification difficult without the use of a SILAP standard.
The third obstacle remains the extraordinary complexity of proteins present in human serum. In standard 2D-LC-MS/MS protocols, proteins are fractionated after trypsin digestion. Human serum contains proteins that are present over a wide dynamic range,16,17
so peptides from abundant proteins can become widely distributed, interfering with identification of lower abundance proteins in many of the fractions collected. One successful approach to improving the number of low abundance proteins identified has been to perform increasing numbers of orthogonal separation steps after tryptic digestion, either serially or in parallel. Some examples include 1D gel-electrophoresis,(18
) N-linked glycopeptide enrichment,(20
) and cysteinyl peptide enrichment.(20
) Immunoaffinity removal of abundant proteins(21
) has also proved to be a robust and reproducible method for studying lower abundance proteins in serum.
Fewer methods have been developed to successfully integrate separation methods at the intact protein level with 2D-LC-MS/MS. IEF of intact proteins is one of the most common methods for separating complex protein mixtures. Historically, this has been performed as the first dimension of 2D gel electrophoresis. Few studies have been reported, however, demonstrating how this technique can be leveraged for sample separation prior to 2D-LC-MS/MS. One study using liquid phase IEF prior to trypsin digestion and reversed-phase 1D-LC-MS/MS suggested the feasibility of this approach.(22
) A similar approach was taken to study plasma and amniotic fluid.(23
) In this study, a proprietary IEF apparatus was used, prior to 1D-LC-MS/MS. However, only 73 and 69 proteins were identified in the respective samples. The power of IEF was suggested by a more comprehensive study, which included solution IEF followed by 1D-gel electrophoresis and LC-MS/MS, and was able to identify over 2000 serum proteins.(24
) With the development of modern immobilized pH gradient (IPG) strips, the reproducibility and resolution of IEF separation has been significantly improved. IPG strip capacity provides another major advantage over other procedures, as up to 3 mg of protein can be readily loaded on an 18 cm strip.(25
) In the current study, we have developed a method combining the use of a SILAP standard with immunoaffinity removal of abundant proteins and IEF-2D-LC-MS/MS analysis to identify a large number of pancreatic cancer associated biomarkers.