Comparative evaluation of peptide desalting methods for salivary proteome analysis

Jehmlich, Nico; Golatowski, Claas; Murr, Annette; Salazar, Gesell; Dhople, Vishnu M.; Hammer, Elke; Völker, Uwe

doi:10.1016/j.cca.2014.04.003

Cited by 17 publications

(10 citation statements)

References 12 publications

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“…Due to the inherently low total protein concentration (i.e., 2.03 mg/mL measured in the development pool, as determined by Bradford assay ), preliminary concentration steps using TCA/acetone, ammonium sulfate, chloroform/methanol, or centrifugal ultrafiltration were compared. In the final method, the salivary proteins were precipitated with trichloroacetic acid/acetone and dithiothreitol, incubated for 15 min on ice, and then washed twice with 100% ice‐cold acetone, as described previously . After decanting the supernatant, the pellet was rehydrated in a solubilization solution for Bradford (1.5 mg/mL measured) and bottom‐up LC/MRM–MS.…”

Section: Methodsmentioning

confidence: 99%

Multiplexed panel of precisely quantified salivary proteins for biomarker assessment

et al. 2016

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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.

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Section: Methodsmentioning

confidence: 99%

Multiplexed panel of precisely quantified salivary proteins for biomarker assessment

et al. 2016

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“…Free amino acids are present at low concentrations, and examples are given in Table 1. At least 400 different proteins have been detected in whole human saliva (18). Over 95% of salivary polypeptides belong to the major salivary protein families including acidic and basic proline-rich proteins, amylases, mucins MUC5B (MG1) and MUC7 (MG2), salivary agglutinin gp340, cystatin, histatins, and statherin (19).…”

Section: The Composition Of Salivamentioning

confidence: 99%

Saliva as the Sole Nutritional Source in the Development of Multispecies Communities in Dental Plaque

Jakubovics

2015

Microbiol Spectr

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Dental plaque is a polymicrobial biofilm that forms on the surfaces of teeth and, if inadequately controlled, can lead to dental caries or periodontitis. Nutrient availability is the fundamental limiting factor for the formation of dental plaque, and for its ability to generate acid and erode dental enamel. Nutrient availability is also critical for bacteria to grow in subgingival biofilms and to initiate periodontitis. Over the early stages of dental plaque formation, micro-organisms acquire nutrients by breaking down complex salivary substrates such as mucins and other glycoproteins. Once dental plaque matures, dietary carbohydrates become more important for supragingival dental plaque, and gingival crevicular fluid forms the major nutrient source for subgingival microorganisms. Many species of oral bacteria do not grow in laboratory monocultures when saliva is the sole nutrient source, and it is now clear that intermicrobial interactions are critical for the development of dental plaque. This chapter aims to provide an overview of the key metabolic requirements of some well-characterized oral bacteria, and the nutrient webs that promote the growth of multispecies communities and underpin the pathogenicity of dental plaque for both dental caries and periodontitis.

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“…Comparisons of lone or combined desalting strategies and sorbent brands were evident in recent research. Four commercially available brands of C18, namely μC18 ZipTip® pipette tips, C18 ZipTip® pipette tips, TopTip C‐18, and OASIS® hydrophilic–lipophilic balanced (HLB) μElution Plate, were compared in terms of their reproducibility and peptide recoveries for tryptic‐digested proteins from saliva samples before LC‐MS/MS determination . All four SPE devices exhibited equal efficiency in desalting and purification of about 340 protein samples.…”

Section: Desaltingmentioning

confidence: 99%

Sample Clean‐up Strategies for ESI Mass Spectrometry Applications in Bottom‐up Proteomics: Trends from 2012 to 2016

2017

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Bottom-up proteomics is a mass spectrometric (MS)-based approach for the characterization of peptides obtained from in-solution protein digestion. MS is favored over other methods for peptide and protein analysis because of its better sensitivity and high throughput. Inorganic ions and surfactants present in the sample or produced during tryptic digestion are detrimental in MS analysis and affect the proteome data, thus sample preparation for removal of these unwanted components has become essential. Here, we review 48 research papers on strategies for removal of salts and surfactants (in particular, SDS) prior to ESI-MS analysis in bottom-up proteomics from 2012 to 2016. The strategies were mostly based on SPE and membrane-based filter-aided sample preparation for salt and SDS removal, respectively. Some known limitations of SPE and filter-aided sample preparation procedures are that they can be time consuming, laborious, and require the use of organic solvents before a concentrated extract suitable for analysis is obtained. The development of faster analytical methods by reducing the sample preparation time and thereby, increasing sample throughput, and in a solvent-less and membrane-less operation, is a significant contribution to proteome research.

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Comparative evaluation of peptide desalting methods for salivary proteome analysis

Cited by 17 publications

References 12 publications

Multiplexed panel of precisely quantified salivary proteins for biomarker assessment

Multiplexed panel of precisely quantified salivary proteins for biomarker assessment

Saliva as the Sole Nutritional Source in the Development of Multispecies Communities in Dental Plaque

Sample Clean‐up Strategies for ESI Mass Spectrometry Applications in Bottom‐up Proteomics: Trends from 2012 to 2016

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