Evaluation of the ALiPHAT method for PC-IDMS and correlation of limits-of-detection with nonpolar surface area

Abstract

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...

Capitalizing on the hydrophobic bias of electrospray ionization through chemical modification in mass spectrometry-based proteomics

“…Based on their experience of increasing the hydrophobicity of nucleic acids through chemical modification, in 2007 the Muddiman group developed a method, termed Augmented Limits of Detection for Peptides with Hydrophobic Alkyl Tags (ALiPHAT), to selectively increase the ESI response of cysteine-containing peptides and proteins ( Figure 2D) [26][27][28][29]. Given that it utilizes hydrophobic analogs on the commonly used cysteine alkylation reagent, iodoacetamide, the ALiPHAT method can be seamlessly integrated into a traditional bottom-up proteomics experiment.…”

“…In a subsequent report, the Muddiman group evaluated a collection of hydrophobic alkylation reagents containing a variety of alkyl and phenyl functionalities [27]. In Modified with permission from [28,29].…”

“…In two other proof-of-concept studies, the Muddiman group applied their library of hydrophobic alkylation reagents into a protein cleavage isotope dilution MS workflow to augment to the quantification of the biomarkers prostate specific antigen [28] and BNP-32 [29]. In both cases, the optimal alkylation reagent was shown to decrease the assay detection limits by twofold and 3.5-fold for the tryptic peptides of prostate specific antigen and BNP-32, respectively.…”

“…In both cases, the optimal alkylation reagent was shown to decrease the assay detection limits by twofold and 3.5-fold for the tryptic peptides of prostate specific antigen and BNP-32, respectively. In these latter two studies, the Muddiman group also applied nonpolar surface area (NPSA) calculations as a method for relating the overall hydrophobicity of the modified peptides to the observed ESI responses [28,29]. Figure 3A shows results for the three peptides evaluated between these two studies.…”

“…In particular, the refinement of existing models, such as NPSA [28,29] or sequence-specific retention calculator [21], will allow for hypothesis-driven synthesis of future reagents and will allow for in silico prediction, rather than empirical determination of each peptide's optimal tag. Indeed, such prediction methods could even be utilized in future studies to determine whether a hydrophilic tag would be of benefit for inherently hydrophobic peptides.…”