Journal of Chromatography B (v.847, #1)

Biomarker discovery by mass spectrometry symposium, May 18–19, 2006 by R. Bischoff; Theo M. Luider; Roel J. Vonk; Ron A. Heeren; Sander Piersma (1-2).

The extraordinary developments made in proteomic technologies in the past decade have enabled investigators to consider designing studies to search for diagnostic and therapeutic biomarkers by scanning complex proteome samples using unbiased methods. The major technology driving these studies is mass spectrometry (MS). The basic premises of most biomarker discovery studies is to use the high data-gathering capabilities of MS to compare biological samples obtained from healthy and disease-afflicted patients and identify proteins that are differentially abundant between the two specimen. To meet the need to compare the abundance of proteins in different samples, a number of quantitative approaches have been developed. In this article, many of these will be described with an emphasis on their advantageous and disadvantageous for the discovery of clinically useful biomarkers.
Keywords: Biomarker discovery; Mass spectrometry; Quantitative proteomics; Targeted quantitation;

SELDI-TOF mass spectra: A view on sources of variation by Martijn Dijkstra; Roel J. Vonk; Ritsert C. Jansen (12-23).
Adequate interpretation of mass spectrometry data can yield valuable biomarkers. However, spectrum interpretation is a complicated task. This paper reviews the various factors that determine a sample's spectrum and demonstrates the role of these factors in the interpretation process. We derive a simulation model that adequately predicts the expected spectrum based on known sample content and, in the reverse mode, obtain an analysis model that adequately fits an observed spectrum based on the hypothesized sources of variation.
Keywords: Mass spectrometry; Quantitative analysis; SELDI; Mixture models; Spectrum analysis;

Direct on-membrane peptide mass fingerprinting with MALDI–MS of tyrosine-phosphorylated proteins detected by immunostaining by Tsuyoshi Nakanishi; Eiji Ando; Masaru Furuta; Susumu Tsunasawa; Osamu Nishimura (24-29).
We have identified tyrosine-phosphorylated proteins on membrane from A-431 human epidermoid carcinoma cells by using detection with anti-phosphotyrosine antibody followed by PMF analysis. In there, on-membrane digestion for these protein spots was carried out on microscale region using chemical inkjet technology and the resulting tryptic digests were directly analyzed by MALDI–TOF MS. Proteins identified by a database search included phosphoproteins that are known to be markedly phosphorylated on tyrosine sites after the cells are treated with epidermal growth factor (EGF). This procedure is a rapid and easily handled approach that enables both detection and identification of phosphoproteins on a single blot membrane.
Keywords: Phosphorylation; Immunodetection; On-membrane digestion; Protein identification; MALDI–TOF MS;

Unravelling in vitro variables of major importance for the outcome of mass spectrometry-based serum proteomics by Mikkel West-Nørager; Christian Dahl Kelstrup; Christian Schou; Estrid V. Høgdall; Claus K. Høgdall; Niels H.H. Heegaard (30-37).
The use of mass spectrometry (MS) for analysing low-molecular weight proteins and peptides from biological fluids has a great, yet not fully realized, potential for biomarker discovery. To prune MS-data as much as possible for non-relevant non-biological variation the development of standardized protocols for handling and processing the samples before MS and adjusting data after MS to compensate for method-induced variability are warranted. This calls for knowledge about how different variables contribute to MS-based proteome analyses. In addition, identification of the peptides involved in pre-analytical variation will be helpful in evaluating the clinical significance of predictive models derived from MS data. Using human sera, extraction by weak cation-exchange magnetic beads, and analysis by MALDI-TOF MS we here evaluated pre-analytical variation and identify peptides involved in this. The influences of humidity, temperature, and time for preparation of sera on spectral changes were evaluated. Also, the reproducibility of the methods and the effect of a baseline correction procedure were examined. Low temperatures, short handling times, and a baseline correction procedure minimize the contribution of artifacts to sample variability as observed by MS. The complement split product C3f and fragments thereof appear to be sensitive indicators of sample handling induced modifications. Other peptides that are indicative of such variability are fibrin and kininogen fragments. Using strict experimental guidelines as well as standardized sample collection procedures it is possible to obtain reproducible peak intensities and positions in serum mass profiling using magnetic bead-based fractionation and MALDI-TOF MS.
Keywords: Proteomics; Mass spectrometry; Serum; In vitro variables; Proteins; Peptides; Variability; MALDI-TOF MS; Humidity; Temperature; Time; Baseline correction; Complement C3f; Magnetic bead-based fractionation; Reproducibility;

Validation of an HIV-1 inactivation protocol that is compatible with intracellular drug analysis by mass spectrometry by Jeroen J.A. van Kampen; Esther J. Verschuren; Peter C. Burgers; Theo M. Luider; Ronald de Groot; Albert D.M.E. Osterhaus; Rob A. Gruters (38-44).
Mass spectrometry is a powerful tool for studying the intracellular pharmacokinetics of antiretroviral drugs. However, the biohazard of HIV-1 calls for a safety protocol for such analyses. To this end, we extracted HIV-1 producing cells with methanol or ethanol at 4 °C. After extraction, no viral infectivity was detected, as shown by a reduction in infectious titers of more than 6 log. In addition, this protocol is compatible with the quantitative analysis of antiretroviral drugs in cell extracts using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Thus, using this protocol, infectious HIV-1 is inactivated and antiretroviral drugs are extracted from cells in a single step.
Keywords: HIV; Infectious; Antiviral; Inactivation; MALDI; Intracellular;

SELDI-TOF-MS of saliva: Methodology and pre-treatment effects by Raymond Schipper; Arnoud Loof; Jolan de Groot; Lucien Harthoorn; Eric Dransfield; Waander van Heerde (45-53).
Interest in saliva as a diagnostic fluid for monitoring general health and for early diagnosis of disease has increased in the last few years. In particular, efforts have focused on the generation of protein maps of saliva using advanced proteomics technology. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a novel high throughput and extremely sensitive proteomic approach that allows protein expression profiling of large sets of complex biological specimens. In this study, large scale profiling of salivary proteins and peptides, ranging from 2 to 100 kDa was demonstrated using SELDI-TOF-MS. Various methodological aspects and pre-analytical variables were analysed with respect to their effects on saliva SELDI-TOF-MS profiling. Results show that chip surface type and sample type (unstimulated versus stimulated) critically affect the amount and composition of detected salivary proteins. Factors that influenced normal saliva protein profiling were matrix composition, sample dilution and binding buffer properties. Delayed processing time experiments show certain new peptides evolving 3 h post-saliva donation, and quantitative analyses indicate relative intensity of other proteins and peptides changing with time. The addition of protease inhibitors partly counteracted the destabilization of certain protein/peptide mass spectra over time suggesting that some proteins in saliva are subject to digestion by intrinsic salivary proteases. SELDI-TOF-MS profiles also changed by varying storage time and storage temperature whereas centrifugation speed and freeze–thaw cycles had minimal impact. In conclusion, SELDI-TOF-MS offers a high throughput platform for saliva protein and peptide profiling, however, (pre-)analytical conditions must be taken into account for valid interpretation of the acquired data.
Keywords: Salivary proteome; Specimen sampling and handling; Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry;

Clinically relevant biomarkers are urgently needed for improving patient diagnosis, risk stratification, prognosis and therapeutic treatments. There is a particularly compelling motivation for identifying protein-based indicators of early-stage disease for more effective interventions. Despite recent progress, the proteomic discovery process remains a daunting challenge due to the sheer heterogeneity and skewed protein abundances in biofluids. Even the most advanced mass spectrometry systems exhibit limiting overall dynamic ranges and sensitivities relative to the needs of modern biomedical applications. To this end, we report the development of a robust, rapid, and reproducible high performance ion-exchange liquid chromatography pre-fractionation method that allows for improved proteomic detection coverage of complex biological specimens using basic tandem mass spectrometry screening procedures. This form of sample simplification prior to global proteomic profiling, which we refer to collectively as ‘fractionomics’, increases the number and diversity of proteins that can be confidently identified in tissue and cell lysates as compared to the straight analysis of unfractionated crude extracts.
Keywords: Proteomics; Biomarker; Sample preparation; Pre-fractionation; HPLC; Ion exchange chromatography; LC–MS; Shotgun sequencing; Tandem mass spectrometry;

In this paper we describe a combination of the mass spectrometric techniques MALDI–TOF/TOF and MALDI–FTMS to identify proteins in complex samples using prespotted MALDI target plates. By this procedure accurate FTMS mass measurements and TOF/TOF data are obtained from the same spot. We have found that this combination of techniques leads to more reliable identification of peptides.
Keywords: Proteomics; FTMS; MALDI–TOF/TOF; Peptide identification;

Depletion of high-abundance proteins from serum by immunoaffinity chromatography: A MALDI-FT-MS study by Lennard J. Dekker; Jan Bosman; Peter C. Burgers; Angelique van Rijswijk; Robert Freije; Theo Luider; Rainer Bischoff (65-69).
Immunodepletion of high-abundance proteins from serum is a widely used initial step in biomarker discovery studies. In the present work we have investigated the reproducibility of the depletion step by comparing 250 serum samples from prostate cancer patients. All samples were depleted on a single immunoaffinity column over a time period of 6 weeks with automated peak detection and fraction collection. Reproducibility in terms of surface area of the depleted serum protein peak at 280 nm was below 7% relative standard deviation (R.S.D.) and the collected volume of the relevant fraction was 0.97 mL (4.5% R.S.D.). Proteins in the depleted serum fraction were subsequently digested with trypsin and analyzed by MALDI-FT-MS. The degree of the depletion of albumin, transferrin and alpha-1-antitrypsin was determined by comparing the intensity of peptide peaks before and after depletion of 11 samples taken at regular time intervals from amongst the 250 depleted, randomized samples. As a positive control we evaluated peaks of apolipoprotein A1 (the most abundant serum protein remaining after depleteion) showing a clear increase in intensity of these peaks in the depleted samples. From this study we conclude that the depletion of the 250 serum samples was complete and reproducible over a period of 6 weeks.
Keywords: Biomarker discovery; Fourier transform mass spectrometry; Immunoaffinity chromatography; Prostate cancer; Proteomics;