September, 2007 (Vol. 3, No. 3) Scientific Updates Recent Publications Collaborative Software Administrative Updates Biomarker Team Spotlight Key Web Pages at FHCRC

Dear ICBC Team Members:

I am happy to announce that the next International Cancer Biomarker Consortium (ICBC) meeting is confirmed for February 20–22, 2008 in Honolulu, Hawaii. There will be a welcoming reception held on Wednesday evening, February 20 and everyone is invited to attend. The scientific meeting will take place on February 21 & 22.

I commend the ICBC team members who have been organizing regional conferences. As a follow-up to successful regional workshops last year, Dr. Myeong-Hee Yu and Prof. Eunok Paek, of the Functional Proteomics Center, are organizing a workshop on Bioinformatics, October 5-6, 2007 at Korea University. Dr. John Luk (HKU) and Dr. Hyang-Sook Yoo (KRIBB) co-chaired a well-attended liver cancer biomarker roundtable discussion on August 25^th at Hong Kong University. Representatives from Hong Kong, Taiwan, China and Korea teams participated. A report on the roundtable discussion will be included in the next newsletter.

Dr. Dick Smith and colleagues at PNNL, have provided us a scientific update on improving low-level plasma protein mass spectrometry-based detection for candidate biomarker discovery and validation. In addition, we have included a link to a list of recent publications from Consortium members.

The team highlight this issue is the colon cancer team led by Drs. Connie Jimenez and Remond Fijneman at VU University Medical Center in Amsterdam.

Best regards,

Lee Hartwell
President and Director
Fred Hutchinson Cancer Research Center

   

Improving low-level plasma protein mass spectrometry-based detection for candidate biomarker discovery and validation
Jason Page, Ryan Kelly, David Camp, and Dick Smith, Pacific Northwest National Laboratory, Richland, WA

Methods. To improve the detection of low abundance proteins for candidate biomarker discovery and validation, particularly in complex biological fluids such as blood plasma, increased sensitivity is desired using mass spectrometry (MS)-based instrumentation. A key current limitation on the sensitivity of electrospray ionization (ESI) MS is due to the fact that many sample molecules in solution are never ionized, and the vast majority of the ions that are created are lost during transmission from atmospheric pressure to the low pressure region of the mass analyzer. Two important technologies, multi-nanoelectrospray emitters and the electrodynamic ion funnel have recently been developed and refined at Pacific Northwest National Laboratory (PNNL) to greatly improve the ionization and transmission efficiency of ESI MS based analyses. Multi-emitter based ESI enables the flow from a single source (typically a liquid chromatography [LC] column) to be divided among an array of emitters (Figure 1). The flow rate delivered to each emitter is thus reduced, allowing the well-documented benefits of nanoelectrospray¹ for both sensitivity and quantitation to be realized for higher flow rate separations. To complement the increased ionization efficiency afforded by multi-ESI, tandem electrodynamic ion funnels have also been developed at PNNL, and shown to greatly improve ion transmission efficiency in the ion source interface.², ³ These technologies have been integrated into a triple quadrupole mass spectrometer for multiple reaction monitoring (MRM) of probable biomarker candidates in blood plasma and show promise for the identification of new species even at low level concentrations.

Application. Adaptation of ion funnel technologies with commercial MS platforms has been shown to provide a significant increase in sensitivity.^{4, 5} Ion funnels replace the inefficient skimmers in the ESI interfaces that conventionally sample the ion/gas jet entering the mass spectrometer with extremely poor ion transmission. In contrast, the ion funnels provide a marked enhancement and allow all of the ions to be captured and focused while a majority of the background gas is pumped out. Implementation of the ion funnel on commercially available Thermo Fisher Scientific mass spectrometers, for example, provided an ~10-fold increase in sensitivity.⁵ The analysis of a cellular lysate before and after installment of the ion funnel showed that sensitivity was enhanced while the high quality separation was maintained (Figure 2). This provided an ~2-fold increase in protein identifications at the lowest sample concentration (30 ng/µL) that was analyzed. Further enhancement of the ion funnel interface has been achieved by adding a second ion funnel in tandem which was operated at a higher pressure.² The higher pressure of the first ion funnel enabled a variety of ion inlet configurations which improved the transmission efficiency and increased the sensitivity. For example, the multiple capillary inlet shown in Figure 1 allows the larger ion currents from the multi-ESI sources to be efficiently captured and transferred to the mass spectrometer.

Combining the tandem ion funnels with arrays of 19 chemically etched fused silica emitters permits both the ionization efficiency and the ion transmission efficiency to be simultaneously improved.^{3, 6} For both infusion-based ESI MS and LC/MS analyses performed using a time-of-flight mass spectrometer, the sensitivity of the multi-ESI sources in combination with the tandem ion funnel was 10-fold higher than that of a single emitter/single ion funnel configuration.⁵ Importantly, peak shape and resolution were preserved for the LC separations, and the signal-to-noise ratios (S/N) were vastly improved. Figure 3 shows several replicate extracted ion chromatograms for a low abundance peptide from a protein that was spiked into plasma and tryptically digested. Not only did sensitivity improve with the multi-ESI source, but S/N improved ~7-fold for the chromatograms obtained using the multi-ESI configuration.⁶

The multi-ESI/multi-capillary inlet source has initially been evaluated for a blood plasma sample using a tandem ion funnel with a modified triple quadrupole mass spectrometer using MRM methods. The increased sensitivity has allowed significantly improved identification and quantitation of low level peptides and proteins. Our plan is to combine these advances with the use of greater selectivity obtained by the incorporation of an ion mobility based FAIMS separation stage to provide both improved sensitivity and selectivity to aid candidate biomarker discovery.

Figure 1.
Multi-ESI/tandem ion funnel configuration for improved ionization and ion transmission efficiency.

		Schematic depiction of the instrument modifications, including a multi-capillary inlet and differentially pumped tandem ion funnels.
		Photograph of an array of 19 emitters positioned in front of a heated multi-capillary inlet.

Figure 2.
LC-MS chromatograms from the analysis of 1 µg samples of a digested cellular lysate obtained using a commercial linear ion trap MS:

		– without an electrodynamic ion funnel interface.
		– with an electrodynamic ion funnel interface.

Figure 3.
Extracted ion chromatograms of the peptide LDQWLCEK from bovine β-lactalbumin spiked into human plasma.

		A multi-emitter ESI source with a multi-capillary inlet and tandem ion funnels was employed for the replicate analyses in (a). The scale bar in the left panel of (a) equals 1 min and applies to all panels.
		A single emitter/single inlet was used with an ion funnel in (b)

References

1. Karas, M.; Bahr, U.; Dulcks, T. Fresenius J. Anal. Chem. 2000, 366, 669-676.
2. Ibrahim, Y.; Tang, K. Q.; Tolmachev, A. V.; Shvartsburg, A. A.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2006, 17, 1299-1305.
3. Kelly, R. T.; Page, J. S.; Tang, K. Q.; Smith, R. D. Anal. Chem. 2007, 79, 4192-4198.
4. Shaffer, S. A.; Prior, D. C.; Anderson, G. A.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1998, 70, 4111-4119.
5. Page, J. S.; Tang, K.; Smith, R. D. Int. J. Mass Spectrom. 2007, 265, 244-250.
6. Kelly, R. T.; Page, J. S.; Zhao, R.; Qian, W.-J.; Mottaz, H. M.; Tang, K.; Smith, R. D. Anal. Chem. 2007, 79, submitted.

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You may access the list of recent publications from Consortium members here.

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None at this time.

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Regional Workshop: Bioinformatics Workshop at Korea University, October 5–6, 2007

Dr. Myeong-Hee Yu and Prof. Eunok Paek of the Functional Proteomics Center (KIST) are hosting a Bioinformatics Workshop October 5–6, 2007 at Korea University in Seoul, Korea. The workshop is full, and registration is now closed. A brief summary about the workshop will be included in the next newsletter.

ICBC Meetings

The next ICBC meeting will be held February 20–22, 2008 in Honolulu, on the island of Oahu, Hawaii. The meeting will take place at the Hyatt Regency Waikiki Resort and Spa. A link to the meeting website is included below. Please note: you must go the Location/Accommodations link to book your hotel room and the Registration link to register for the meeting.
www.fhcrc.org/science/international_biomarker/meetings/2008/feb

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Project Title:		Colorectal Cancer Biomarker Discovery
Cancer Site(s):		Colorectal
Team Coordinator:		Gerrit A. MEIJER, Ph.D
Principal Investigator(s):		Connie R. JIMENEZ, Ph.D. Remond J.A. FIJNEMAN, Ph.D.
Participating Institutions:		VU University Medical Center (VUmc), Amsterdam, the Netherlands
Clinical Samples:		Serum/plasma, CRC adenoma and carcinoma tissue
Mouse Model(s):		APC-mutant mice
Technical Approaches:		HTP MALDI-TOF-MS-based profiling NanoLC-LTQFTMS Sub-proteomes (Low Mw-, glyco-, surface-, nuclear- proteomes) Label-free protein quantitation iTRAQ quantitation MALDI Tissue Profiling/Imaging Immuno-MS 1D/2DGE Comparative Genomic Hybridization (array-CGH profiling) mRNA-expression profiling (Agilent platform) RNAi Global (genome-wide siRNA kd library, Dharmacon) Tissue microarray / immunohistochemistry Antibody generation (phage display library) Bead-based Multiplexing Assay System

Brief Description of Project:

Research of the Tumor Profiling Unit of the VUmc Cancer Center Amsterdam, focuses on translational and basic studies aiming to reduce the high death rates from gastrointestinal cancer with a focus on colorectal cancer (CRC). This includes secondary prevention of CRC, and optimized therapy selection for CRC. Concerning secondary prevention, we aim to 1. identify biomarkers that discriminate pre-malignant lesions with low potential to progress into carcinomas ('low-risk' adenomas) from 'high-risk' adenomas (tumor profiling by arrayCGH, transcriptomics, and proteomics), and 2. to identify individuals at increased risk (genetic predisposition) for sporadic cancer (blood profiling by transcriptomics and proteomics). Concerning optimized therapy selection, we aim to follow the response to therapy by analyzing blood samples of CRC patients before and after treatment (blood-profiling by proteomics).

The current program of the VUmc-CCA team will focus on the application of mass spectrometry (MS)-based proteomics to the discovery and quantitation of protein biomarkers for colorectal cancer. This team builds on ample clinical, genomics and proteomics expertise and new state-of-the-art infrastructure in MS and proteomics at VUmc. Three major application development areas are 1. high-throughput peptide profiling of body fluids by magnetic bead-based sample processing off-line coupled to MALDI-TOF-MS, 2. label-free peptide profiling of tissue and body fluid subproteomes by nanoLC-LTQFTMS and 3. the selective enrichment and quantitative analysis of candidate cancer-related genes identified by genomics/transcriptomics/proteomics by targeted tandem mass spectrometry methods. The VUmc-CCA team will be supported by a rapidly growing informatics group, which will develop and implement (open-source) data analysis tools for label-free quantitation of LC-MS datasets. After the discovery and initial validation of candidate CRC biomarkers, our final goal is to develop blood tests for risk assessment and early detection of CRC.

Team Members and Expertise:

Gerrit A. MEIJER, M.D., Ph.D.
Dr. Gerrit A. Meijer is CRC Team Coordinator, head of the Tumor Profiling Unit.

Connie R. JIMENEZ, Ph.D.
Dr. Connie R. Jimenez is head of the recently established OncoProteomics Laboratory of the Cancer Center Amsterdam at the VUmc, and coordinates the VUmc-clinical proteomics activities in the context of the Center for Medical Systems Biology, a national genomics center of excellence. In 1994, at the Vrije Universiteit in Amsterdam, she pioneered the use of MALDI-TOF-MS for differential display of neuropeptides in single cells and, in 1997, for semi-quantitative neuropeptide profiling directly in tissue homogenates. Since 1997 (while at UCSF in the Burlingame lab), she has worked in the field of proteomics and has ample expertise in the handling of complex biological samples (whole tissue, cells, organelles and more recently body fluids). In 2000, Dr. Jimenez initiated and is still coordinating the Netherlands Proteomics Platform. She is member of the general council of the European Proteomics Association (EuPA) and participates in the operation committee EuPA-HUPO interactions. Moreover, she is member of the biomarker discovery committee of the HUPO-Brain project.

Remond J.A. FIJNEMAN, Ph.D.
Dr. Remond J.A. Fijneman is Research Associate of the Tumor Profiling Unit. He has a strong background in mouse models for human cancer, which he used to dissect the genetic complexity of predisposition to sporadic cancer, and to investigate molecular interactions between epithelial tumor cells and hematopoietic (stromal) cells. Currently, he aims to 1. identify a blood-derived RNA-expression profile indicative of CRC risk that can be used as a pre-likelihood test for CRC screening, and 2. to identify biomarkers for colorectal cancer that can be used for molecular imaging, a project that is carried out together with a Ph.D. student (Meike de Wit, M.Sc.), in collaboration with Philips Research.

Beatriz CARVALHO, Ph.D
Dr. Beatriz Carvalho is Research Associate of the Tumor Profiling Unit. She has a strong background in Comparative Genomic Hybridization and expression arrays, using large series of colon adenoma and carcinoma samples. Based on these data, she currently aims to identify gene(s) that are responsible for adenoma-to-carcinoma progression, a project that is carried out together a Ph.D. student (Anke Hardebol, M.Sc.).

Sander PIERSMA, Ph.D.
Dr. Sander Piersma is Research Associate of the OncoProteomics Laboratory. Dr. Piersma is a biochemist with a background in protein isolation, purification and characterization. Since 2002, he is active in the area of imaging mass spectrometry as well as LC-MS. In 2005, Dr. Piersma published the first mass spectrometry imaging at subcellular resolution. In the OPL, he is leading the nanoLC-LTQFTMS workflows as well as direct tissue profiling/imaging by MALDI-TOF-MS, both with a focus on native peptides and proteins.

Jaco KNOL, Ph.D.
Dr. Jaco Knol is a Ph.D. level research technician of the OncoProteomics Laboratory with ample expertise in molecular biology and biochemistry. Dr. Knol is performing all MALDI-TOF/TOF workflows with a focus on method development for the analysis of tissue (focused on the cell nucleus) and biological fluids using MALDI-TOF-MS for biomarker discovery.

Thang PHAM, Ph.D.
Dr. Thang Pham is Research Associate of the OncoProteomics Laboratory. Dr. Pham is a computer scientist with expertise in pattern recognition. The aim is to implement robust data pre-processing routines that will be combined with multi-variate statistical approaches (supervised and non-supervised) to create reliable statistical models that are validated and robust enough for patient classification and biomarker discovery. Dr. Thang works in close collaboration with data analysis experts in the Depts of Clinical Epidemiology and Biostatistics (Prof. Dr. Maarten Boers), Statistics (Vrije Universiteit Amsterdam, Prof.Dr. Aad van der Vaart), and Computer Science (Vrije Universteit Amsterdam, Dr. Elena Marchiori).

Bauke YLSTRA, Ph.D.
Dr. Bauke Ylstra is head of the VUmc Micro-Array Facility. The VUMC MA-facility offers high resolution array CGH as well as expression arrays (Agilent platform). The facility has several coworkers, including a statistician, Dr. Mark van der Wiel, specialized in 'omics' data analysis. See further.

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