Journal of Chromatography B, 902 (2012) 70–77 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B jo u r n al hom epage: www.elsevier.com/locate/chromb Quantification of HPLC-separated peptides and proteins by spectrofluorimetric detection of native fluorescence and mass spectrometry Suraj Saraswat, Bruce Snyder 1 , Dragan Isailovic ∗ Department of Chemistry, University of Toledo, Toledo, OH, USA a r t i c l e i n f o Article history: Received 14 February 2012 Accepted 17 June 2012 Available online 4 July 2012 Keywords: Quantification Peptides Proteins HPLC Native fluorescence ESI-MS a b s t r a c t Due to relatively low reproducibility of the ionization and differences when using buffers as mobile phases, the quantitative analysis by electrospray ionization mass spectrometry (ESI-MS) can be often challenging. In the present study, the native fluorescence of phenylalanine, tyrosine, and tryptophan was investigated as an improvement tool for the analytical quantification of peptides and proteins by HPLC–ESI-MS. Natively fluorescent amino acids as well as peptides, proteins, and protein digests were successfully separated by HPLC, and quantified with a spectrofluorimetric detector and ESI-MS. The two detectors were connected in series and enabled the sequential measurements of the fluorescence inten- sities as well as the measurements of the ion signals and mass spectral characterization of separated polypeptides. Fluorescence detector provided better linearity and repeatability of quantification than mass spectrometer, and similar limits of detection for most of biomolecules analyzed. The fluorescence signal was linear over 3–4 orders of magnitude with limits of detection in picomole or high femtomole range, depending on nature and number of natively fluorescent amino acid residues present in the ana- lyzed polypeptides. Hence, native fluorescence of phenylalanine, tyrosine, and tryptophan can be used as a label-free methodology to facilitate quantification of peptides and proteins by LC–ESI-MS. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Mass spectrometry is widely applied for identification and structural characterization of proteins and their post-translational modifications. Most of protein MS analyses are conducted by ESI and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry [1,2]. To analyze complex proteomic samples, bottom-up and top-down MS techniques were developed. In the bottom-up approach, a protein mixture is subjected to enzymatic digestion, and HPLC–MS is then used for separation of digest peptides and protein identification [3,4]. Reversed-phase HPLC (RP- HPLC) in combination with ESI-MS is most commonly used in such applications. In top-down approach, a proteomic sample is sepa- rated and individual proteins are investigated directly by MS/MS [3,4]. In addition to qualitative structural analysis, LC–ESI-MS can be used for quantification of proteins using labeling and label-free techniques. Isotopic labeling is often used in the case of mass ∗ Corresponding author at: Department of Chemistry, University of Toledo, MS 602, 2801 W. Bancroft St, Toledo, OH 43606, USA. Tel.: +1 419 530 5523; fax: +1 419 530 4033. E-mail address: Dragan.Isailovic@utoledo.edu (D. Isailovic). 1 Current address: Emmanuel Christian School, 4607 Laskey Rd., Toledo, OH 43623, USA. spectrometric quantification of peptides and proteins [5–7]. Quan- titative analysis can be done using isotopic labeling by amino acids in cell culture (SILAC) [5], isotope-coded affinity tags (ICAT) [6], and isobaric tags for relative and absolute quantification (iTRAQ) [7]. For example, cysteines that are isotopically labeled by ICAT reagents can be used for quantification of proteins based on the presence of doublets in the mass spectra corresponding to “heavy” and “light” isotopes [6]. These procedures require expensive isotopic labels and extensive sample preparation protocols. In addition, label-free methodologies have also been reported for protein quantification in biological samples [8,9]. In all of these quantification experiments, the mass spectrometers can operate in single stage acquisition mode [10] and single ion recording (SIR) mode [11], or in multi- ple stage acquisition modes such as low-energy collision-induced dissociation tandem mass spectrometry (CID–MS/MS) and multi- ple reactions monitoring (MRM) [12–14]. Commonly, HPLC enables separation while an ESI mass spectrometer is used for structural characterization and quantification of polypeptides. However, LC–ESI-MS has its own quantification drawbacks such as ionization suppression and irreproducible ionization especially when different buffers are used as mobile phases [15]. Differences among MS instruments (i.e., variability of ion sources and mass ana- lyzers) also complicate comparative quantification. All these factors can affect accuracy and reproducibility of the MS quantification. The addition of another independent detection method could be useful 1570-0232/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2012.06.018