Activated PSCs are known to secrete ECM proteins such as collagens I, III,
and IV, and fibronectin10,11. This matrix then serves to promote cell proliferation
and migration to heal wounded areas. Chronic activation, as seen in chronic pancreatitis or PDAC, results in the accumulation of fibrotic tissue. PSCs are also known to produce factors that break down ECM, which may play a role in promoting invasion. Indeed, PDAC lymph node metastases were observed to contain stroma and PSCs (Figure 8). In addition, 24 ECM proteins and proteases and 40 proteins associated with cell motility and vasculature development were identified.
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Figure 8. IHC staining of PDAC lymph node metastasis. PSCs are visualized by the
expression of markers -SMA (A) and vimentin (B) and the absence of CD31(C).
Positive staining is in brown.
ANXA2, a phospholipid binding protein, is believed to play a role in angiogenesis, invasion, and metastasis, and its expression has been best
described in colon cancer106,107, ANXA2 has also been reported to serve as a
receptor for plasminogen108 and as such may play a role in the plasminogen
activator pathway The urokinase plasminogen activator pathway is believed
to play a role in cancer cell invasion in the pancreas92,94 and includes the
proteins MMP2 and SERPINE1, along with PLAU. SERPINI2 is a protein
specific to the pancreas and to breast cancer stroma109,110. The function of
SERPINI2 is poorly understood, however its sequence identifies it as a serine protease inhibitor in the same family as SERPINE1, suggesting a role in the plasminogen activator pathway, as well. LGALS3BP, a protein expressed in
advanced gastric cancer111, binds the receptor galectin-3, a protein highly
expressed in PDAC metastases112. LGALS3BP modulates cell-integrin
interactions to either promote or inhibit cell attachment113. NME1 has been
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cancer114,115. In vitro studies have shown that NME1 suppresses tumor
metastatic potential and negatively regulates cell migration, possibly via
regulation of integrin expression or trafficking116-118. TGFBI is a secreted
RGD-containing protein and has anti-adhesive properties119,120. TGFBI is
upregulated in renal cell carcinoma and esophageal adenocarcinoma121,122,
and has been reported to inhibit cell attachment in vitro119.
Finally, UCHL1 and EZR are believed to enhance invasion. UCHL1 is thought to modify cell morphology through modulation of the Akt-signaling
pathway123. EZR is a mediator of cell-integrin interactions and high
expression of EZR has been associated with prostate and pancreatic cancer
progression124,125. By organizing the interaction between the cytoskeleton
and the cell membrane EZR regulates cell adhesion, migration and
invasion126. Furthermore, EZR has been found to be essential to the
metastatic potential of some cell lines127,128. Taken together, our results
demonstrate PSCs are an important source of proteins that regulate ECM remodeling, cell motility and invasion.
The proteins discussed here are a sampling of the many secreted proteins identified from the activated PSC secretome. It is clear that the PSC secretome is an important source of proteins that regulate proliferation, inflammation, and ECM remodeling as well as cell motility and invasion. Characterization of the activated PSC secretome is an important first step in elucidating the complex role of PSCs in PDAC progression. These results lay the foundation for future studies, for example, characterizing the proteins
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involved in paracrine signaling with PDAC cells. Further studies would determine whether secreted PSC proteins identified here can serve as biomarkers of PDAC progression or potential new therapeutic targets.
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3 Chapter 3 – Development of an LC-MS method for the relative
quantitation of potential biomarker proteins
For submission as:
Wehr AY, Blair IA, Yu KH. Relative quantitation of serum pancreatic cancer biomarker proteins by stable isotope dilution liquid chromatography-mass spectrometry. JPR 2011
3.1 Abstract
An innovative multiplexed LC-MRM/MS-based assay for rapidly measuring a large number of disease specific protein biomarkers in human serum has been developed. This approach is based on stable isotope dilution methodology in order to reliably quantitate candidate protein biomarkers. Human serum was diluted using a SILAP standard prepared from the secretome of pancreatic cell lines, subjected to immunoaffinity removal of the most highly abundant proteins, trypsin digested, and analyzed by LC- MRM/MS. The method was found to be precise, linear, and specific for relative quantitation of 72 proteins when analyte response was normalized to the relevant internal standard (IS) from the SILAP. The method made it possible to determine statistically different concentrations for 3 proteins in control and PDAC patient samples. This method proves the feasibility of using SILAP dilution with MRM analysis of tryptic peptides as a means to compare changes in the concentration of candidate protein biomarkers in human serum.
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3.2 Introduction
Many methods have been developed to characterize differential proteomes, resulting in vast lists of candidate biomarker proteins which await
quantitation in human samples for validation48-55. ELISA is the most common
method used for the quantitation of proteins in serum due to its high sensitivity and specificity. However, this method becomes expensive and labor intensive when applied to a large number of proteins and samples. Furthermore, high quality monoclonal antibodies are not available for the vast majority of candidate biomarkers identified. As a result, most
biomarker validation studies are limited to single or several proteins56-59.
Stable isotope dilution with detection by MS has been the mainstay of
bioanalytical analyses for the past two decades69-71, due to both the
specificity and sensitivity with which this methodology can measure the concentration of small molecules. With developments in MS technology, such an approach has provided the speed and sensitivity to allow for detection of
low abundance proteins in serum72,73.
Compared with small molecule LC-MS, the analysis of proteins has some specific challenges. Intact proteins can be difficult to separate on traditional LC columns, especially those designed to work at the decreased flow rates required to achieve high sensitivity microflow electrospray. However, the peptides that result from tryptic digestion behave much more like small molecules and are easier to separate by RP LC with MS-compatible mobile phases. Tryptic peptides terminating in lysine and arginine are also
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amenable to multiple charging by ESI. This makes it possible to generate
products ions of a lower charge state and higher m/z than their
corresponding precursor ions, which helps to minimize background noise, a key concern when working in a complex matrix like human serum.
The protein load in human serum poses another concern. The LC columns used with microflow ESI/MS are limited in the amount of protein that can be loaded onto the column. The concentration range of proteins in human
serum spans over 10 orders of magnitude74 and 99% of the protein by mass
in serum is made up of 22 highly abundant proteins such as albumin and
immunoglobulins75. SISCAPA has been developed as a system to purify
proteins of interest from the biological matrix76. However, antibodies specific
to each protein of interest must first be developed, and thus is limiting in a similar fashion to ELISA. Commercially available immunoaffinity columns can be used to remove these proteins from the serum, reducing the protein concentration and effectively increasing the loading capacity of the LC
column77-79.
The processes of digestion and immunoaffinity removal of abundant proteins are not without drawbacks and both have been associated with high variability which can confound studies intended to compare the concentration
of an analyte across different samples83,129. Dilution with a stable isotope
labeled IS is commonly used in bioanalytical assays to correct for losses during sample preparation or variability in chromatographic separation or
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means to normalize proteomic profiling, a methodology that was termed
SILAP50,73,81. The current study represents a novel implementation of SILAP
for the relative quantitation of a panel of potential serum protein biomarkers. Stable isotope labeled secreted proteins from human pancreatic cell lines in culture were used as the SILAP standard. The development and validation of a quantitative method, using relevant IS from the SILAP to normalize the analyte response in human serum has now been completed.