ORIGINAL PAPER HPLC-CHIP coupled to a triple quadrupole mass spectrometer for carbonic anhydrase II quantification in human serum Luciano Callipo & Patrizia Foglia & Riccardo Gubbiotti & Roberto Samperi & Aldo Laganà Received: 17 November 2008 / Revised: 23 February 2009 / Accepted: 11 March 2009 / Published online: 24 March 2009 # Springer-Verlag 2009 Abstract A method for carbonic anhydrase II (CA II) absolute quantification in human serum is presented. This method is based on high-performance liquid chromatogra- phy (HPLC)-Chip microfluidic device incorporating a nanoelectrospray source interfaced to a triple quadrupole mass spectrometer. The fraction containing CA II was isolated by preparative reversed-phase HPLC, and peptides obtained from the tryptic digest of the protein mixture were separated by the HPLC-Chip system. The multiple-reaction monitoring acquisition mode of a selected suitable CA II peptide and peptide internal standard allowed the selective and sensitive determination of a CA II. Absolute recovery of the method was 52±12%, while analytical recovery was 81±10%. For the eight samples analyzed, the matrix effect was found to be only -14±6%. A comparison among three regression lines type which were obtained by external calibration, matrix-matched calibration, and standard addi- tion method, respectively, demonstrated that the first one is adequate in obtaining good accuracy and precision. Method quantification limit for CA II in serum was estimated to be 2 fmol/mL. CA II mean concentration in sera from eight healthy subjects was found to be 56 pmol/mL (relative standard deviation 24%). Keywords Liquid chromatography-tandem mass spectrometry . Chip . Triple quadrupole . Peptides . Protein absolute quantification Introduction Over the recent years, proteomics has moved from an effort to identify the proteins of biological systems to targeted strategy aiming to identify key proteins, such as biomarkers that can provide reliable diagnostic and prognostic indica- tors of disease progression or treatment effects. However, till now, this tool has been only partly exploited. Rapid immunotest development and clinical validation are time- consuming and costly steps which restrict the new biomarker adoption in clinical praxis. Due to its flexibility, mass spectrometry (MS) may constitute a more accessible analytical tool for preclinical protein biomarker studies. However, it remains difficult to identify biomarkers using conventional proteomic approaches because of their low abundance [1]. In addition to the initial identification of phenotypic expression and protein characterization, a key parameter in proteomic analysis is the ability to quantify proteins of interest. To date, a majority of the quantitative proteomic analyses have been performed using stable isotope labeling strategies such as isotope coded affinity tag [2], isobaric tags for relative and absolute quantitation (iTRAQ™)[3], stable isotope labeling by amino acids in cell culture [4], 18 O labeling [5, 6], and stable isotope standards with capture by anti-peptide antibodies [7, 8]. These methodologies require complex, time-consuming sample preparation and can be relatively expensive [9]. One of the most powerful and used proteomics approaches for low-abundance protein identification involves the ultrasmall-bore liquid chromatography coupled to nanoelectrospray MS (nano-LC-MS). Recently, it has been presented a microfabricated approach to nano-LC that integrates all the components on a single LC Chip, eliminating the need for conventional LC connections [10]. LC-Chip-MS technology allows by itself multi- Anal Bioanal Chem (2009) 394:811–820 DOI 10.1007/s00216-009-2752-6 L. Callipo : P. Foglia : R. Gubbiotti : R. Samperi : A. Laganà (*) Department of Chemistry, Sapienza University of Rome, Box nº 34, Roma 62, Piazzale Aldo Moro 5, 00185 Rome, Italy e-mail: aldo.lagana@uniroma1.it