Abstract

Recent technological advances in instrumentation and sample preparation have raised the attention towards MS-based targeted protein immunoassays as attractive alternative to classical ELISAs [1]. The Mass Spectrometry ImmunoAssay (MSIA(TM)) approach utilizes the MSIA D.A.R.T.'s technology which comprises a molecular trapping micro-column contained within a pipette housing. A capture antibody is immobilized onto the micro-column and the antigen containing sample is applied by repetitive pipetting cycles. After washing and elution steps, the target antigen can be either directly injected into mass spectrometers or subjected to enzymatic digestion for peptide markers detection. The combination of, immunoaffinity capture and mass spectrometry provide complementary advantages: the first enables a specific binding of the target molecule from a complex matrix using the immobilized antibody with its consequent selective enrichment on the micro-column and the prospective to fully automate the procedure; MS instead provides an unambiguous detection, as antigen signals are observed at characteristic m/z values in the mass spectrum, overcoming common issues of immunoassays, such as false positives.So far, MSIA technology has found mainly applications in clinical fields where the complexity of biofluids, such as blood plasma and serum, impairs the sensitivity, robustness and throughput of routine MS-based protein detection [2 and reference thereof].In the present investigation, MSIA approach was applied to the food safety field. Notably, the applicability of a MSIA based workflow was tested for the detection of residual egg proteins in wine. Egg-derived products, in various commercial preparations, are commonly utilized in winemaking thanks to the ability to interact and promote precipitation of wine polyphenols and other undesirable compounds [3]. However, any residues of egg white proteins remaining in wine could represent a risk for allergic consumers. In this scenario, the development of analytical approaches for the detection of egg proteins might open new perspectives for the producers, that might spot the real risk associated to certain procedures where allergens are likely to remain as residues. Till now, no official method for the determination of fining agent proteins is prescribed.Here, the feasibility of the MSIA approach for the detection of such residues in white wines was investigated, combining the semi-automated immunoaffinity enrichment with selective reaction monitoring (SRM) detection of specific peptide markers. The micro-columns in the MSIA D.A.R.T's were chemically modified to bind a polyclonal antibody commercially available to specifically recognize native ovalbumin (OVA), the most abundant protein in the egg based fining agent typically used for wine clarification. As a proof of concept, the MSIA D.A.R.T's were employed with the Finnpipette(TM) Novus i Multichannel Electronic Pipette which processes up to 12 samples simultane

Autore Pugliese

• Monaci Linda , Pilolli Rosa

Tutti gli autori

• R. Pilolli; A. Di Palo; R. Chaudari; L. Monaci

Titolo volume/Rivista

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Anno di pubblicazione

2015

ISSN

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ISBN

978-88-909328-6-1

Numero di citazioni Wos

Nessuna citazione

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Numero di citazioni Scopus

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Settori ERC

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Codici ASJC

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