**2.6 Identification and characterization of 2-D protein spots**

#### **2.6.1 Sequence data from 2-D gels**

2-DE has the virtually unique capability of simultaneously displaying several hundred gene products. 2-D gels are an ideal starting point for protein chemical identification and characterization. Peptide mass fingerprint or sequence data can be derived following 2-DE with mass spectrometry or amino acid sequence analysis (Eckerskorn *et al.* 1988, Ducret *et al.* 1996). The sensitivity of currently available instruments makes 2-DE an efficient "preparative" analytical method. Most current protein identification depends on mass spectrometry of proteins excised from gels or blots.

#### **2.6.2 Integration of image analysis with automated spot cutting**

Image analysis software obtains quantitative and qualitative information about the proteins in a sample, and stores the information in files, which may also contain additional annotations. The ProteomeWorks™ spot cutter expands the capabilities of proteome labs by integrating PDQuest™ image analysis software. The image analysis files acquired by PDQuest direct automated spot cutting. Excised protein spots are deposited into microtiter plates ready for further automated processing. PDQuest software tracks the protein spots through spot cutting and protein identification. Downstream protein spot identifications are 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 unique identification.

#### **2.6.3 Automated protein digestion**

374 Gel Electrophoresis – Advanced Techniques

series that are not present in the reference gel are added manually so that the reference gel

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

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

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

2-DE has the virtually unique capability of simultaneously displaying several hundred gene products. 2-D gels are an ideal starting point for protein chemical identification and characterization. Peptide mass fingerprint or sequence data can be derived following 2-DE with mass spectrometry or amino acid sequence analysis (Eckerskorn *et al.* 1988, Ducret *et al.* 1996). The sensitivity of currently available instruments makes 2-DE an efficient "preparative" analytical method. Most current protein identification depends on mass

Image analysis software obtains quantitative and qualitative information about the proteins in a sample, and stores the information in files, which may also contain additional annotations. The ProteomeWorks™ spot cutter expands the capabilities of proteome labs by integrating PDQuest™ image analysis software. The image analysis files acquired by PDQuest direct automated spot cutting. Excised protein spots are deposited into microtiter plates ready for further automated processing. PDQuest software tracks the protein spots through spot cutting and protein identification. Downstream protein spot identifications are

will include all proteins of a gel series.

comparison to the reference gel.

**2.6.1 Sequence data from 2-D gels** 

**2.5.7 Data analysis** 

tests are available.

require verification, as does automatic spot detection.

**2.6 Identification and characterization of 2-D protein spots** 

**2.6.2 Integration of image analysis with automated spot cutting** 

spectrometry of proteins excised from gels or blots.

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 eliminates a significant bottleneck for high-throughput protein identification.

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 with the Mass*PREP* clean air enclosure.

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

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

Two Dimensional Gel Electrophoresis in Cancer Proteomics 377

To confirm the presence of isoforms of CRT, they performed two-dimensional Western blotting with two different antibodies: monoclonal antibody FMC75, which was produced against recombinant human CRT; and a polyclonal antibody that was produced using synthesized peptides of the human CRT COOH terminus (amino acids 388–400) as an immunogen. On Western blots with anti- COOH-terminus antibody, only one of the two spots was visualized, whereas both spots became visible on blots incubated with FMC75 (Fig. 3). One was the same as the 55-kDa (pI 4.3) spot, and the other had an apparent molecular mass of 40 kDa and pI of 4.5. This lower molecular- mass spot had the same NH2 terminal amino acid sequence as amino acids 1–10 of mature human CRT as shown by amino acid sequencing. Therefore, they suggested that the higher-molecular-mass spot was the full-length form and the other spot was a cleaved form that is truncated elsewhere in the COOH domain. Production of the full-length CRT in cancer tissue was increased compared with in healthy tissue, but the spots for cleaved CRT in cancerous and healthy urothelium

had intensities that were similar and were reproducible on all silverstained 2DE gels.

Fig. 1. Silver-stained images of analytical narrow-pH-range 2DE gels of proteins from bladder cancer. (A), pH 4.0–5.0; (B), pH 4.5–5.5; (C), pH 5.0–6.0; (D), pH 5.5–6.7. Arrows indicate spots (U-1 to U-15) containing higher amounts of protein. Spot U-2, ~55 kDa and pI 4.3, was confirmed to be CRT by a peptide mass fingerprinting method and NH2-terminal amino acid sequencing. *(Source: Susumu Kageyama et al., 2004 Clinical Chemistry, 50: 857–866)*

COOH terminus antibody is appropriate as a tumor marker.

Subsequently they tried to confirm whether anti-COOH terminus antibody binds to molecules other than full lengt1h CRT and performed immuno-precipitations (Fig. 4). The Western blot of the immuno-precipitate extracted from cancer tissue revealed only one band, and they concluded that anti-COOH-terminus antibody binds specifically to fulllength CRT of ~55 kDa. They therefore judged that full-length CRT recognized by anti-

from spot cutting to protein digestion to MS analysis and image annotation is reduced by over 50% compared to manual processing of gel samples, with a corresponding reduction in error. All of the instrumentation and software in this process is part of the integrated ProteomeWorks system, a set of powerful tools for proteomic analysis.
