Electrospray ionization (ESI) has been an indispensable ion generation technique for mass spectrometric analysis of biopolymers such as intact proteins and protein digests operated at atmospheric pressure. Since its advent in 1998, atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) quickly became a popular alternative for the analysis of peptides. While AP-MALDI sources typically share the same vacuum interface and ion transmission hardware with ESI, it is generally found that ESI is superior in detection sensitivity. In this thesis, we describe a home-built AP-MALDI source coupled to a commercial 7.0 Tesla Fourier Transform Ion cyclotron Resonance Mass Spectrometer (FT ICR MS). A method based on solid-phase extraction and elution with surface-functionalized diamond nanocrystals, which we referred to as “SPEED” that not only streamlines AP-MALDI mass spectrometric analyses of peptides and other small biomolecules under typical operational conditions, but also outruns ESI in ultimate detectable concentration by at least one order of magnitude. The application of home built AP-MALDI source coupled with FTICR-MS was used for the Collision-induced fragmentation study of L-arginine (Arg), ω-NG-monomethylarginine (MMA), ω-NG,NG-asymmetric dimethylarginine (aDMA) and ω-NG,NG’-symmetric dimethylarginine (sDMA). Admixing of nitrocellulose with the matrix reduces significantly the interfering matrix peaks in the resulting mass spectra. Tandem mass spectrometric (MS2 and MS3) methods could easily distinguish the isobaric molecules aDMA, sDMA and other intrinsic interfering molecules originating from a biological matrix. Among a total of 44 fragment ions generated from the two isomeric DMAs, 5 were uniquely produced exclusively from just one of them that allowed their differentiation. The unsurpassed advantage of high resolution and mass accuracy of FTICR MS enabled us to identify fragmentation channels that yield fragment ions differing in mass by only ~0.01 Da. The sub-ppm mass accuracies facilitated proposing chemical structures for all the fragments observed. Many hereto unreported isobaric fragments from the methylated arginines were identified for the first time. Application of tandem mass method of distinguishing these isobaric molecules was demonstrated with human urine. Protocols were developed with commercially available solid phase extraction (SPE) products for extracting arginines and its methylated derivatives from urine samples. Without any derivatization, selected pairs of MS/MS fragment intensities were used for quantitating the relative abundances of these molecules in the urine samples. The urinary aDMA to sDMA ratio in healthy individuals was found to be 0.82 +/- 0.17, in good agreement with most reported results.
Electrospray ionization (ESI) has been an indispensable ion generation technique for mass spectrometric analysis of biopolymers such as intact proteins and protein digests operated at atmospheric pressure. Since its advent in 1998, atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) quickly became a popular alternative for the analysis of peptides. While AP-MALDI sources typically share the same vacuum interface and ion transmission hardware with ESI, it is generally found that ESI is superior in detection sensitivity. In this thesis, we describe a home-built AP-MALDI source coupled to a commercial 7.0 Tesla Fourier Transform Ion cyclotron Resonance Mass Spectrometer (FT ICR MS). A method based on solid-phase extraction and elution with surface-functionalized diamond nanocrystals, which we referred to as “SPEED” that not only streamlines AP-MALDI mass spectrometric analyses of peptides and other small biomolecules under typical operational conditions, but also outruns ESI in ultimate detectable concentration by at least one order of magnitude. The application of home built AP-MALDI source coupled with FTICR-MS was used for the Collision-induced fragmentation study of L-arginine (Arg), ω-NG-monomethylarginine (MMA), ω-NG,NG-asymmetric dimethylarginine (aDMA) and ω-NG,NG’-symmetric dimethylarginine (sDMA). Admixing of nitrocellulose with the matrix reduces significantly the interfering matrix peaks in the resulting mass spectra. Tandem mass spectrometric (MS2 and MS3) methods could easily distinguish the isobaric molecules aDMA, sDMA and other intrinsic interfering molecules originating from a biological matrix. Among a total of 44 fragment ions generated from the two isomeric DMAs, 5 were uniquely produced exclusively from just one of them that allowed their differentiation. The unsurpassed advantage of high resolution and mass accuracy of FTICR MS enabled us to identify fragmentation channels that yield fragment ions differing in mass by only ~0.01 Da. The sub-ppm mass accuracies facilitated proposing chemical structures for all the fragments observed. Many hereto unreported isobaric fragments from the methylated arginines were identified for the first time. Application of tandem mass method of distinguishing these isobaric molecules was demonstrated with human urine. Protocols were developed with commercially available solid phase extraction (SPE) products for extracting arginines and its methylated derivatives from urine samples. Without any derivatization, selected pairs of MS/MS fragment intensities were used for quantitating the relative abundances of these molecules in the urine samples. The urinary aDMA to sDMA ratio in healthy individuals was found to be 0.82 +/- 0.17, in good agreement with most reported results.