Multiplexed panel of precisely quantified salivary proteins for biomarker assessment.

An increasingly popular "absolute" quantitative technique involves the SRM or MRM approach with stable isotope-labeled standards (SIS). Using this approach, many proteins in human plasma/serum have been quantified for biomarker assessment and disease stratification. Due to the complexity of plasma and the invasive nature of its collection, alternative biosamples are currently being explored. Here, we present the broadest panel of multiplexed MRM assays with SIS peptides for saliva proteins developed to date. The validated panel consists of 158 candidate human saliva protein biomarkers, inferred from 244 interference-free peptides. The resulting concentrations were reproducibly quantified over a 6 order-of-magnitude concentration range (from 218 µg/mL to 88 pg/mL; average CVs of 12% over analytical triplicates). All concentrations were determined from reverse standard curves, which were generated using a constant concentration of endogenous material with varying concentrations of spiked-in SIS peptides. The large-scale screening of the soluble and membrane-associated proteins contained within the 158-plex assay could present new opportunities for biomarker assessment and clinical diagnostics.

A comparison of MS/MS-based, stable-isotope-labeled, quantitation performance on ESI-quadrupole TOF and MALDI-TOF/TOF mass spectrometers.

The peptide-based quantitation accuracy and precision of LC-ESI (QSTAR Elite) and LC-MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ-labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC-MALDI spectra. The average protein sequence coverages for LC-ESI and LC-MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ-based expression ratios determined by ProteinPilot from the 57 467 ESI-MS/MS and 26 085 MALDI-MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7-6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC-ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC-MALDI iTRAQ ratios were rejected. Re-analysis of an archived LC-MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS-based peptide quantitation performance of offline LC-MALDI was comparable with on-line LC-ESI, which required threefold less time. However, offline LC-MALDI allows the re-analysis of archived HPLC-separated samples.

Current trends in quantitative proteomics.

It was inevitable that as soon as mass spectrometrists were able to tell biologists which proteins were in their samples, the next question would be how much of these proteins were present. This has turned out to be a much more challenging question. In this review, we describe the multiple ways that mass spectrometry has attempted to address this issue, both for relative quantitation and for absolute quantitation of proteins. There is no single method that will work for every problem or for every sample. What we present here is a variety of techniques, with guidelines that we hope will assist the researcher in selecting the most appropriate technique for the particular biological problem that needs to be addressed. We need to emphasize that this is a very active area of proteomics research-new quantitative methods are continuously being introduced and some 'pitfalls' of older methods are just being discovered. However, even though there is no perfect technique--and a better technique may be developed tomorrow--valuable information on biomarkers and pathways can be obtained using these currently available methods.