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In this review, the author concentrated more on questions that cast doubt …
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- Mass Spectrometry as a Diagnostic and a Cancer Biomarker Discovery Tool
Where should we go from here? In Table V, I summarize some open questions related to this technology. These questions have been posted before (46). Further progress will depend on providing answers after careful experimentation. I would also make some suggestions related to future publications and experiments that need to be done with this technology.
- Because the technology for positively identifying the discriminatory proteins/peptides is now readily available, future investigations should report the identity of at least some of these peptides and connections made to previous publications associating these proteins with cancer.
- Future investigations should include internal controls. For example, for common cancers, where classical tumor markers exist (Table I) and their concentrations in the tested samples are known, one goal should be the identification of these biomarkers for validation purposes. Other controls (e.g. exogenously added peptides) should be included and their peak amplitudes used to correct for other peak amplitudes in different experiments.
- If investigations include "training" and "test" sets, the interpretation algorithm should be tested with an independent series of samples (preferably obtained from another institution) at least 3 months later to validate its robustness.
- When feasible, it will be useful to apply different bioinformatic algorithms on the same set of data. Do different algorithms produce similar outputs (e.g. the same discriminatory peaks and diagnostic sensitivity/specificity) when the same input data are used?
- It is imperative that the actual sensitivity of SELDI-TOF mass spectrometry, as it applies to serum analysis, is carefully evaluated. At the same time, it should be investigated if the provided information is quantitative, semiquantitative, or qualitative. Experiments to examine this could include:
- a. Spiking of serum samples with synthetic peptides 30–50 amino acids long (molecular mass 3,000–5,000 Da). This could be used to establish the detection limit of these peptides with serum as matrix on various protein chips, and also to establish the quantitative nature of the measurement by constructing calibration curves in a serum matrix.
- b. Another way of establishing the detection limit of the method would be to select groups of serum samples with known tumor marker concentration (e.g. free PSA of molecular mass around 30,000 Da and 1-antichymotrypsin-PSA with molecular mass of 100,000 Da). The quantification of free and total PSA in serum is straightforward using ELISA technologies. Groups of serum samples with free or total PSA concentration, e.g. in the range of 0.5, 2, 10, 50, 200, and 2,000 µg/liter, could be run with the SELDI-TOF protocols to establish 1) the detection limit and 2) if the amplitude of the peaks is quantitatively related to the PSA concentration.
- It will be important to establish in controlled experiments if the discriminatory peptides identified in serum are actually produced in vivo or ex vivo. Samples from the same patients should be obtained with or without proteinase inhibitors and processed in various ways, as described by Marshall et al. (59), to examine if the discriminatory peaks originate by the action of proteases after the blood is drawn.
- Studies should be performed to establish the differences in proteomic patterns between plasma and serum and examine the effects of lipemia, length of storage, freeze-thaw cycles, menstrual cycle, age, nutritional status, drug ingestion, sex, race, etc. on such patterns. Without knowledge of the effects of these, and possibly other parameters, on the proteomic patterns generated by MALDI-TOF, the interpretations will be questionable.
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