Dustin C. Frost

Comparison of Two-Dimensional Fractionation Techniques for Shotgun Proteomics

Two-dimensional (2D) fractionation is a commonly used tool to increase dynamic range and proteome coverage for bottom-up, shotgun proteomics. However, there are few reports comparing the relative separation efficiencies of 2D methodologies using low-microgram sample quantities. In order to systematically evaluate 2D separation techniques, we fractionated microgram quantities of E. coli protein extract by seven different methods. The first dimension of separation was performed with either reversed-phase high-pressure liquid chromatography (RP-HPLC), gel electrophoresis (SDS-PAGE), or strong cation exchange (SCX-HPLC). The second dimension consisted of a standard reversed-phase capillary HPLC coupled to an electrospray ionization quadrupole time-of-flight mass spectrometer for tandem mass spectrometric analysis. The overall performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The protein-level RP-HPLC and the high-pH RP-HPLC peptide-level separations performed the best, identifying 281 and 266 proteins, respectively. The online pH variance SCX and the SDS-PAGE returned modest performances with 178 and 139 proteins identified, respectively. The offline SCX had the worst performance with 81 proteins identified. We also examined various chromatographic factors that contribute to separation efficiency, including resolving power, orthogonality, and sample loss.

High-Resolution Enabled 12-Plex DiLeu Isobaric Tags for Quantitative Proteomics

Multiplex isobaric tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ)) are a valuable tool for high-throughput mass spectrometry based quantitative proteomics. We have developed our own multiplex isobaric tags, DiLeu, that feature quantitative performance on par with commercial offerings but can be readily synthesized in-house as a cost-effective alternative. In this work, we achieve a 3-fold increase in the multiplexing capacity of the DiLeu reagent without increasing structural complexity by exploiting mass defects that arise from selective incorporation of 13C, 15N, and 2H stable isotopes in the reporter group. The inclusion of eight new reporter isotopologues that differ in mass from the existing four reporters by intervals of 6 mDa yields a 12-plex isobaric set that preserves the synthetic simplicity and quantitative performance of the original implementation. We show that the new reporter variants can be baseline-resolved in high-resolution higher-energy C-trap dissociation (HCD) spectra, and we demonstrate accurate 12-plex quantitation of a DiLeu-labeled Saccharomyces cerevisiae lysate digest via high-resolution nano liquid chromatography–tandem mass spectrometry (nanoLC–MS2) analysis on an Orbitrap Elite mass spectrometer.

Recent advances in mass spectrometry-based glycoproteomics.

Protein glycosylation plays fundamental roles in many biological processes as one of the most common, and the most complex, posttranslational modification. Alterations in glycosylation profile are now known to be associated with many diseases. As a result, the discovery and detailed characterization of glycoprotein disease biomarkers is a primary interest of biomedical research. Advances in mass spectrometry (MS)-based glycoproteomics and glycomics are increasingly enabling qualitative and quantitative approaches for site-specific structural analysis of protein glycosylation. While the complexity presented by glycan heterogeneity and the wide dynamic range of clinically relevant samples like plasma, serum, cerebrospinal fluid, and tissue make comprehensive analyses of the glycoproteome a challenging task, the ongoing efforts into the development of glycoprotein enrichment, enzymatic digestion, and separation strategies combined with novel quantitative MS methodologies have greatly improved analytical sensitivity, specificity, and throughput. This review summarizes current MS-based glycoproteomics approaches and highlights recent advances in its application to cancer biomarker and neurodegenerative disease research.

Mass Defect-Based N,N-Dimethyl Leucine Labels for Quantitative Proteomics and Amine Metabolomics of Pancreatic Cancer Cells

Mass spectrometry-based stable isotope labeling has become a key technology for protein and small-molecule analyses. We developed a multiplexed quantification method for simultaneous proteomics and amine metabolomics analyses via nano reversed-phase liquid chromatography-tandem mass spectrometry (nanoRPLC-MS/MS), called mass defect-based N,N-dimethyl leucine (mdDiLeu) labeling. The duplex mdDiLeu reagents were custom-synthesized with a mass difference of 20.5 mDa, arising from the subtle variation in nuclear binding energy between the two DiLeu isotopologues. Optimal MS resolving powers were determined to be 240K for labeled peptides and 120K for labeled metabolites on the Orbitrap Fusion Lumos instrument. The mdDiLeu labeling does not suffer from precursor interference and dynamic range compression, providing excellent accuracy for MS1-centric quantification. Quantitative information is only revealed at high MS resolution without increasing spectrum complexity and overlapping isotope distribution. Chromatographic performance of polar metabolites was dramatically improved by mdDiLeu labeling with modified hydrophobicity, enhanced ionization efficiency, and picomole levels of detection limits. Paralleled proteomics and amine metabolomics analyses using mdDiLeu were systematically evaluated and then applied to pancreatic cancer cells.

Development and characterization of novel 8‐plex DiLeu isobaric labels for quantitative proteomics and peptidomics

RATIONALE Relative quantification of proteins via their enzymatically digested peptide products determines disease biomarker candidate lists in discovery studies. Isobaric label-based strategies using TMT and iTRAQ allow for up to 10 samples to be multiplexed in one experiment, but their expense limits their use. The demand for cost-effective tagging reagents capable of multiplexing many samples led us to develop an 8-plex version of our isobaric labeling reagent, DiLeu. METHODS The original 4-plex DiLeu reagent was extended to an 8-plex set by coupling isotopic variants of dimethylated leucine to an alanine balance group designed to offset the increasing mass of the labels reporter group. Tryptic peptides from a single protein digest, a protein mixture digest, and Saccharomyces cerevisiae lysate digest were labeled with 8-plex DiLeu and analyzed via nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS(2) ) on a Q-Exactive Orbitrap mass spectrometer. Characteristics of 8-plex DiLeu-labeled peptides, including quantitative accuracy and fragmentation, were examined. RESULTS An 8-plex set of DiLeu reagents with 1 Da spaced reporters was synthesized at a yield of 36%. The average cost to label eight 100 µg peptide samples was calculated to be approximately

High-Throughput Quantitative Proteomics Enabled by Mass Defect-Based 12-Plex DiLeu Isobaric Tags

15. Normalized collision energy tests on the Q-Exactive revealed that a higher-energy collisional dissociation value of 27 generated the optimum number of high-quality spectral matches. Relative quantification of DiLeu-labeled peptides yielded normalized median ratios accurate to within 12% of their expected values. CONCLUSIONS Cost-effective 8-plex DiLeu reagents can be synthesized and applied to relative peptide and protein quantification. These labels increase the multiplexing capacity of our previous 4-plex implementation without requiring high-resolution instrumentation to resolve reporter ion signals.

Quantitative Glycomic Analysis by Mass-Defect-Based Dimethyl Pyrimidinyl Ornithine (DiPyrO) Tags and High-Resolution Mass Spectrometry

Isobaric labeling has become a popular technique for high-throughput, mass spectrometry (MS)-based relative quantification of peptides and proteins. However, widespread use of the approach for large-scale proteomics applications has been limited by the high cost of commercial isobaric tags. To address this, we have developed our own N,N-dimethyl leucine (DiLeu) multiplex isobaric tags as a cost-effective alternative that can be synthesized with ease using readily available isotopic reagents. When paired with high-resolution tandem mass (MS(n)) acquisition, mass defect-based DiLeu isobaric tags allow relative quantification of up to twelve samples in a single liquid chromatography (LC)-MS(2) experiment. Herein, we present detailed methods for synthesis of 12-plex DiLeu isobaric tags, labeling of complex protein digest samples, analysis by high-resolution nanoLC-MS(n), and processing of acquired data.

Increased N,N-Dimethyl Leucine Isobaric Tag Multiplexing by a Combined Precursor Isotopic Labeling and Isobaric Tagging Approach

We recently developed a novel amine-reactive mass-defect-based chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), for quantitative proteomic analysis at the MS1 level. In this work, we further extend the application of the DiPyrO tag, which provides amine group reactivity, optical detection capability, and improved electrospray sensitivity, to quantify N-linked glycans enzymatically released from glycoproteins in the glycosylamine form. Duplex DiPyrO tags that differ in mass by 45.3 mDa were used to label the glycosylamine moieties of freshly released N-glycosylamines from glycoprotein standards and human serum proteins. We demonstrate that both MALDI-LTQ-Orbitrap and nano-HILIC LC/MS/MS Fusion Lumos Orbitrap platforms are capable of resolving the singly or multiply charged N-glycans labeled with mass-defect DiPyrO tags. Dynamic range of quantification, based on MS1 peak intensities, was evaluated across 2 orders of magnitude. With optimized N-glycan release conditions, glycosylamine labeling conditions, and MS acquisition parameters, the N-glycan profiles and abundances in human serum proteins of cancer patients before and after chemotherapy were compared. Moreover, this study also opens a door for using well-developed amine-reactive tags for relative quantification of glycans, which could be widely applied.

Mass Defect-Based Dimethyl Pyrimidinyl Ornithine (DiPyrO) Tags for Multiplex Quantitative Proteomics

Multiplex isobaric tags have become valuable tools for high-throughput quantitative analysis of complex biological samples in discovery-based proteomics studies. Hybrid labeling strategies that pair stable isotope mass difference labeling with multiplex isobaric tag-based quantification further facilitate these studies by greatly increasing multiplexing capability. In this work, we present a cost-effective chemical labeling approach that couples duplex stable isotope dimethyl labeling with our custom 12-plex N,N-dimethyl leucine (DiLeu) isobaric tags in a combined precursor isotopic labeling and isobaric tagging (cPILOT) strategy that is compatible with a wide variety of biological samples and permits 24-plex quantification in a single LC-MS/MS experiment. We demonstrate the utility of the DiLeu cPILOT approach by labeling yeast digests and performing proof-of-principle quantification experiments on the Orbitrap Fusion Lumos.

Biomaterials differentially regulate Src kinases and phosphoinositide 3-kinase-γ in polymorphonuclear leukocyte primary and tertiary granule release

We have developed a novel amine-reactive mass defect-based chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), that is compact in size, is suitable for various biological samples, and enables highly multiplexed quantification of peptides at the MS1 level without increasing mass spectral complexity. The DiPyrO tag structure incorporates heavy isotopes in a variety of configurations to impart as much as 45.3 mDa or as little as 5.8 mDa per tag between labeled peptides. Notably, peptides containing lysine are labeled with two tags, doubling the imparted mass defect to up to 90.6 mDa for the duplex tags and effectively reducing the resolving power requirement compared to previously reported mass defect-based quantification approaches. This permits current and previous generation LTQ-Orbitrap platforms to perform confident quantitative analyses of two DiPyrO-labeled samples at 100K resolving power, whereas 3-plex and 6-plex quantifications are possible at 240K and 480K resolving powers, respectively. In this work, we discuss the design and synthesis of the DiPyrO tag, characterize its effect on labeled proteome analysis by nanoLC-MS2, and demonstrate proof-of-principle applications of the duplex and triplex tags for quantitative proteomics using high-resolution MS acquisition on the Orbitrap Elite and Orbitrap Fusion Lumos.