Analysis Workflow

Sample volume will be determined by the amount of candidate biomarkers in CRC patient samples. This will be established using the following method: Two types of affinity selectors have been useful in the affinity chromatographic isolation of cancer marker glycoproteins, lectins and antibodies. Unfortunately, it is not known which is best for colorectal cancer. Before launching the analysis of several hundred colorectal cancer patient samples, we will do a preliminary study on ten CRC patients to see which reagents best recognize CRC glycoproteins. The reference sample will be a pooled plasma sample from several hundred normal individuals. Antibodies will be used that target the S-Lewis-X and Lewis-X antigens, respectively. Five lectins will be examined. Column packing materials with immobilized selectors will be made with avidin-biotin affinity.

The glycoproteomics workflow is outlined in the table below.

Step1	Affinity select specific glycoforms of glycoproteins from a 0.1 to 0.5 mL sample using a 0.46 X 5 cm chromatography column
Step2	Elute glycoproteins from the affinity column with an acidic mobile phase
Step3	Separate the affinity selected glycoproteins by reversed phase chromatography (RPC) on a 0.21 X 5 cm column using a mobile phase gradient from 0.1% trifluoroacetic acid (TFA) to 0.1% TFA containing 60% acetonitrile (ACN)
Step4	Collect fractions from the RPC column
Step5	Evaporate the collected fractions to dryness with a Speed Vac
Step6	Add trypsin solution to the dried fractions and digesting. After trypsin digestion, primary amine groups on peptide fragments of proteins will be derivatized with a stable isotope labeling agent that codes peptides in CRC patient and non-cancer control samples according to sample origin. This sample will be mixed in equal volume with a control sample treated in identical fashion through the first six steps and differentially coded with a labeling agent of identical structure to that used on the CRC sample, but different mass. The concentration of all peptides in CRC patient samples relative to controls will be established by the isotope ratio of the isotopomeric peptides derived from the mass spectra
Step7	Desalt the digested fractions with ZipTips and eluting the peptides with 60% ACN from the ZipTip sorbent onto an XCT PLUS ION TRAP plate
Step8	Analyze the peptide fractions by XCT PLUS MS/MS mode
Step9	Process the mass spectral data

Instruments

The Shimadzu 2D LC system is used for efficient and effective sample pre-treatment. Click here to see Fred Rengier in his laboratory with the the Shmiadzu 2D HPLC and attached computer processors and user interface equipment.

The sample analysis uses Agilent's chip cube coupled the XCT PLUS ESI ion trap to deliver sensitive MS analysis of peptides for quantitation and MS/MS analysis of peptides for database searching.

Data Processing and Generated Datasets

Data processing will be achieved in two ways. The first will be to transfer the raw MS/MS data containing m/z values and signal intensity to the statistical models and pattern recognition group. The relative amount of peptides will be judged directly from their signal intensity in spectra. The second mode of data processing will be to identify the individual peptides obtained from RPC fractions and their protein parent. This will be done using the MASCOT, Sorcerer or Spectrum Mill search engines.

The XCT PLUS ESI ion trap in LC-MS mode generates ".D" files during instrument sample analysis. For peptide quantification, Bruker's CompassXport software is used to convert the XCT PLUS ".D" files to Level 1 LC-MS mzXML files. The mzXML file generated by the XCT PLUS in LC-MS mode has more detailed m/z information than the XCT PLUS run in LC-MS-MS mode. The LC-MS mzXML file can be used as input to the Proteomics Discovery Pipeline for data mining. [You must be logged in to access the Proteomics Discovery Pipeline link.]


The mzXML files will also be used by the Cancer Care Engineering statistical modeling group for integrative mathematical modeling.

For protein identification, the XCT PLUS LC-MS-MS generated ".D" files are converted to either

• mzXML Level 2 LC-MS-MS format file used for analysis by Sorcerer software
• PKL format file generated by DataExtractor, a component of Agilent's Spectrum Mill software
• MGF, the MASCOT generated format file used for MASCOT database searching.

These formats produce peak lists and fragmentation patterns which are matched against databases to identify proteins. Users can access the mzXML and MGF files converted from the D format, as use them as input to other identification software or databases. The PLK file is not accessible after conversion from the D format.

In general, the LC-MS and LC-MS-MS phases are separate runs of the XCT PLUS. The LC-MS phase includes hundreds of samples, and the LC-MS-MS phase includes only a handful of the original sample group.