**2.5.4 Computer-assisted image analysis of 2-D electrophoretic gels**

Computer-assisted image analysis software is an indispensable tool for the evaluation of complex 2-D gels. It allows:


Image analysis systems deliver error-fee comprehensive qualitative and quantitative data from a large number of 2-D gels (Miller 1989). PDQuest software from Bio-Rad is a popular analysis tool. Gel analysis of digitized gel images includes spot detection, spot quantitation, gel comparison, and statistical analysis. PDQuest software has the further advantage of seamless integration with any of Bio-Rad's image acquisition instruments, as well as the ability to control the ProteomeWorks™ spot cutter. The advanced annotation feature can be used to label spots with text, URL links, document links, or mass spectrometry data.

#### **2.5.5 Spot detection and spot quantitation**

Before the software automatically detects the protein spots of a 2-D gel, the raw image data are corrected and the gel background is subtracted. The process is executed with simple menus and "wizards." PDQuest software models protein spots mathematically as 3-D Gaussian distributions and uses the models to determine absorption maxima. This enables automatic detection a resolution of merged spots. Following this procedure, spot intensities are obtained by integration of the Gaussian function. The mathematical description of the spots is used both for data reduction and for increasing evaluation speed, since reevaluation of data after an image change takes only fractions of a second. The hit rate of automatic spot detection is highly dependent on the quality of the 2-D gels. Correction capabilities of PDQuest software can be used to add undetected spots to the list of spots or to delete spots that arise from gel artifacts.

#### **2.5.6 Gel comparison**

The next step in 2-D gel evaluation is the identification of proteins that are present in all gels of a series. This task is made difficult primarily because of inherent irreproducibility in gels, which affects the positions of spots within a gel series. Gel analysis software must detect minor shifts in individual spot position within the gel series. Many software packages for automatic gel comparison are created with the assumption that the relative positions of spots are altered only slightly relative to each other, and allocate the spots on this basis. Prior to automatic gel comparison, PDQuest software selects the best 2-D gel of a gel series as a reference or standard gel and compares all other 2-D gels to this gel. Proteins in a gel

Two Dimensional Gel Electrophoresis in Cancer Proteomics 375

generally obtained from peptide mass fingerprint analysis using mass spectrometry. The ProteomeWorks spot cutter is a precision instrument with a small benchtop footprint. It is fully automatic to increase throughput and minimize the amount of hands-on time spent excising protein spots. The spot cutter individually excises even overlapping spots for

The ProteomeWorks spot cutter eliminates the first of two bottlenecks for excision and enzymatic digestion of protein spots. Driven by PDQuest software, it enables automated spot excision and deposition of cut gel spots into microtiter plate wells. Isolated proteins from the gel pieces are then digested to release peptides for detailed sequence analysis by mass spectrometry, leading to protein identification. Excised gel spots can be robotically destained, chemically modified (reduced and alkylated), and digested in preparation for either MALDI-TOF-MS or ESI-MS with the Micromass Mass*PREP* station. Each process is executed under fully automated software control with a range of standard protocols enabling high throughput and flexibility. Manual protein digestion is a tedious, time-consuming process that is subject to variability and keratin contamination. Automation of this process with the Mass*PREP* station

Operational features of the Mass*PREP* station include a variable temperature control for optimized reduction, alkylation, and digestion of proteins, and onboard cooling capabilities for reagents and peptide digests to ensure reproducible digestion results. The station employs a variety of sample cleaning technologies (Mass*PREP* targets and Millipore ZipTip pipet tips) to prepare peptide digests prior to automated deposition of samples onto a M@LDI or Mass*PREP* target plate. Contamination of peptide samples is also minimized

Peptide mass fingerprinting of protein digest products using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) provides an ideal method for protein identification when samples have been separated by 2-D PAGE. The M@LDI HT is one of a new generation of networked "2-D gel-MS" analyzers for highthroughput protein identification. M@LDI HT is the primary MS data acquisition device of the ProteomeWorks system, and features a fully automated target plate auto-changer for increased throughput. Networking enables distribution of data capture, protein assignment, and result presentation functions of ProteinLynx Global *SERVER* software within a secure clientserver architecture, maximizing computing power to quickly identify proteins. The M@LDI HT enables automated acquisition of optimized mass spectra and the derivation of monoisotopic peptide mass fingerprint information. Interrogation of multiple FASTA databases using Global *SERVER* software following capture of MS results provides rapid identification of proteins that fit the samples' peptide mass fingerprint, along with a confidence score indicating the validity of the identifications. Following MS identification, peptide mass fingerprint spectra and all of the identification results are available through electronic reports. In addition, protein identification results are seamlessly integrated with the gel image in PDQuest software. Using this system, the working time to process data

eliminates a significant bottleneck for high-throughput protein identification.

**2.6.4 Rapid, high-throughput protein identification by MALDI-TOF-MS** 

unique identification.

**2.6.3 Automated protein digestion** 

with the Mass*PREP* clean air enclosure.

series that are not present in the reference gel are added manually so that the reference gel will include all proteins of a gel series.

Before the software can detect and document matching of different spots, a number of landmarks, or identical spots in the gel series, must be manually identified. The landmarking tool speeds the process by making "best guess" assignments of landmark spots to images in the gel series. With PDQuest software, it is possible to simultaneously display up to 100 enlarged details of 2-D gels on the screen. This simultaneous display of all 2-D gels of a test series enables rapid and error-free determination of the fixed points. Using the landmarks, the image analysis software first attempts to compare all spots lying very near these fixed points and then uses the matched spots as starting points for further comparisons. Thus, the entire gel surface is systematically investigated for the presence or absence of matching spots in a gel series. The results of the automatic gel comparison require verification, as does automatic spot detection.

Two tools assist this verification process in PDQuest: Either identical protein spots are labeled with matching letters and allocated section by section, or the deviations in the spot positions of a particular 2-D gel can be displayed as lines that show spot shifts in comparison to the reference gel.

### **2.5.7 Data analysis**

With PDQuest software, all gels of an experiment are viewed as a unit. To compare gels from different experiments, the reference images are compared. In such comparisons, each spot is automatically assigned a number so that identical spots have identical numbers. Experimental data can also be analyzed statistically both parametric and nonparametric tests are available.
