Target Discovery and Validation Reviews and Protocols

Pancreatic Cancer 61
1.4. Mass Spectrometric Analysis of Proteins
Initial sample collection and preparation are crucial for obtaining optimal
results for most analytical techniques, including mass spectrometry analysis of
proteins. Despite the high sensitivity of current mass spectrometers (routine
analysis at femtomole level), great care should be taken to avoid degradation of
the sample because of the high abundance of naturally occurring proteases present
in pancreatic juice and pancreatic tissue. General guidelines and protocols regarding
sample handling, initial preparation, and fractionation by one-dimensional
(1D) electrophoresis, staining, tryptic digestion, nano-LC-MS/MS analysis,
database searches, and validation of data are provided in Subheading 3.3.8.
It should be emphasized that these protocols are general protocols that should
be modified if more specific analysis is needed. It is very important not to contaminate
the sample with, e.g., keratin (hair and skin). In addition, universal
precautions must be followed during handling of the specimens.
1.5. Molecular Targets Identified by Large-Scale Transcriptomic
and Proteomic Analysis of Human Pancreatic Cancers
The large-scale analyses of human pancreatic cancers by using techniques
described above (Subheadings 1.2.–1.4.) has led to the identification of nearly
200 differentially overexpressed transcripts and a similar number of differentially
overexpressed proteins (15–18). These molecules have immediate translational
relevance in terms of patient care, harboring the potential for application
in imaging studies, targeted therapies, and early detection. As the ensuing discussion
highlights, such translational work is actively underway. For example,
the transcripts prostate stem cell antigen (PSCA) and mesothelin were
identified by Argani et al. (19,20) as being differentially overexpressed in
human pancreatic cancers by SAGE analysis (19,20). Because both PSCA and
mesothelin are membranous proteins, they can be used for specific targeting of
imaging agents to pancreatic cancers in vivo (35) or as a target for monoclonal
antibody therapy by using humanized antibodies (see http://www.agensys.
com). Mesothelin also has been used as a target for immunotoxin therapy with
a conjugated anti-mesothelin antibody (36). Most recently, it also has been
reported that mesothelin is a target antigen against which a host immunologic
reaction is mounted by the cytotoxic T-cell repertoire in human pancreatic cancer
patients (37); thus, it is currently being used in the preparation of pancreatic
cancer vaccines. In addition to therapeutic applications for pancreatic cancer,
PSCA and mesothelin also are secreted proteins; therefore, one can envision
quantitative assessment of these molecules in pancreatic juice samples for early
detection of pancreatic cancer in at-risk patients (38). Claudin 4 is an overexpressed
cell surface protein in human pancreatic cancers identified using an