No abstract

A method employing protein cleavage isotope dilution mass spectrometry (PC-IDMS) was developed for quantification of C-reactive protein (CRP) in human plasma. This method was completed without the use of immuno-affinity chromatography or size exclusion chromatography, as previous mass spectrometric methods for the quantification of CRP have employed. A total of 110 human plasma samples were analyzed with PC-IDMS via 1-D nano LC-MS/MS using a 30 minute gradient with a triple quadrupole mass spectrometer operated in selective reaction monitoring (SRM) mode. The results from this newly developed method were compared to results generated from an enzymelinked immunosorbent assay (ELISA) performed by an independent CLIA certified laboratory. The comparison of these results generated a R 2 = 0.9708 which indicates successful quantification of CRP from human plasma utilizing the methodology described herein. Interestingly, the PC-IDMS method provided concentration values that were ~10X the concentration reported by the ELISA method, which demonstrated that the method of detection is an important consideration when determining reference ranges of a particular analyte. In addition, data is shown that illustrates that as epithelial ovarian cancer (EOC) progresses from stage I to stage IV, mean levels of CRP increase.

Affinity ligand HWRGWV has demonstrated the ability to isolate human immunoglobulin G (hIgG) from mammalian cell culture media. The ligand specifically binds hIgG through its Fc portion. This work shows that deglycosylation of hIgG has no influence on its binding to the HWRGWV ligand and the ligand does not compete with Protein A or Protein G in binding hIgG. It is suggested by the mass spectrometry (MS) data and docking simulation that HWRGWV binds to the pFc portion of hIgG and interacts with the amino acids in the loop Ser383–Asn389 (SNGQPEN) located in the CH3 domain. Subsequent modeling has suggested a possible three-dimensional minimized solution structure for the interaction of hIgG and the HWRGWV ligand. The results support the fact that a peptide as small as a hexamer can have specific interactions with large proteins such as hIgG.

Fourier transform ion cyclotron resonance mass spectrometry has the ability to achieve unprecedented mass measurement accuracy (MMA); MMA is one of the most significant attributes of mass spectrometric measurements as it affords extraordinary molecular specificity. However, due to space-charge effects, the achievable MMA significantly depends on the total number of ions trapped in the ICR cell for a particular measurement. Even through the use of automatic gain control (AGC), the total ion population is not constant between spectra. Multiple linear regression calibration in conjunction with AGC is utilized in these experiments to formally account for the differences in total ion population in the ICR cell between the external calibration spectra and experimental spectra. This ability allows for the extension of dynamic range of the instrument while allowing mean MMA values to remain less than 1 ppm. In addition, multiple linear regression calibration is used to account for both differences in total ion population in the ICR cell as well as relative ion abundance of a given species, which also affords mean MMA values at the parts-per-billion level.

PC-IDMS experiments for two peptides, laminin nonapeptide and the N-terminal tryptic peptide of prostate specific antigen, were performed utilizing a variety of alkylating reagents. These experiments were conducted to investigate how hydrophobicity influences the limitsof-detection (LOD) by altering their electrospray ionization response. Nonpolar surface areas were calculated for both peptides and all alkylating reagents to provide an estimate of the hydrophobicity of the differently alkylated peptides. Decreases in LOD by 2-fold were observed for both peptides between the best and worst performing combination of alkylating reagent. However, while an increase in hydrophobicity was found to aid in decreasing LOD to an extent, beyond a certain hydrophobicity, we observed a decrease. (J Am Soc Mass Spectrom 2009, 20, 2006 -2012 ) © 2009 American Society for Mass SpectrometryA bsolute quantification of a protein from its digestion products dates back to Barr et al. [1]. A combination of proteolysis and MS for absolute quantification of a European Community Bureau of Reference (BCR) certified apolipoprotein A-1 standard that was proteolyzed with trypsin and quantified with a stable isotope labeled internal standard peptide with LC-flow-FAB MS/MS [1]. Their results demonstrated that the use of protein cleavage coupled with isotope dilution mass spectrometry (PC-IDMS) was valid methodology for the standardization of measurements of particular proteins in a clinical environment. PC-IDMS performs best when protein cleavage is complete, as this produces a 1:1 M ratio between the initial intact protein and the peptide or peptides to be analyzed. The quantification using IDMS is based on the ratio of the response of the labeled internal standard peptide to that of the unlabeled peptide resulting from the digestion of the particular protein of interest. IDMS has been utilized on a range of different analytes for almost 40 y and still maintains its utility for the quantification of several different molecules [2].The process of utilizing chemical tags to investigate the quantitative potential of mass spectrometry has been studied previously. [7,8] in addition to the iTRAQ [9] reagent. Other tagging strategies for relative quantification include C-terminal tagging [10] and phosphorylation site modification [11]. Several chemical tagging strategies are described in recent reviews [12][13][14]. The basic idea of exploiting chemical derivatization of analytes with mass spectrometry has been thoroughly studies and produced promising results.In a typical bottom-up proteomics experiment, step reduction and alkylation of cysteines is completed before enzymatic or chemical digestion to eliminate to protein's tertiary structure [15,16]. The alkylation step is the ideal choice for modification of protein/peptide chemical structure since the reaction is fairly straightforward and would eliminate the need for additional labeling steps. Several techniques for chemical tagging utilizing alkylation are summarized in two reviews by Leitner...