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.

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 reverse phase high pressure liquid chromatography (RP-HPLC), gel electrophoresis (SDS-PAGE), or strong cation exchange (SCX-HPLC). The second dimension consisted of a standard reverse phase capillary HPLC coupled to an electrospray ionization quadrupole time-offlight mass spectrometer (ESI-QTOF MS) 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 on-line pH variance SCX and the SDS-PAGE returned modest performances with 178 and 139 proteins identified, respectively. The off-line SCX had the worst performance with 81 proteins identified. We also examined various chromatographic factors which contribute to separation efficiency, including resolving power, orthogonality, and sample loss.

A pronounced asymmetry in the recirculation from blood to lymph of resting small recirculating T lymphocytes is described. When 51Cr-labeled small T-recirculating lymphocytes (TRL) from intestinal lymph were infused intravenously their relative recovery in intestinal lymph was about twice that in nodal lymph. In contrast, the relative recovery in nodal lymph of 51Cr-labeled nodal TRL was twice that in intestinal lymph. Intestinal TRL migrated in large numbers through the small intestine. Nodal TRL did not. It is proposed that the pool of recirculating small T lymphocytes consists of two major subdivisions, an intestinal pool and a nodal pool. The nodal circulation comprises small TRL which traverse PCV in all lymph nodes (LN) but not the small intestine. The intestinal circulation comprises small TRL which do not traverse PCV in LN, but which do recirculate through the small intestine from which they pass via afferent lymphatics to the mesenteric LN and subsequently via the thoracic duct into the blood. It is suggested that the intestinal circulation is present in the fetus and that its initial development is independent of extrinsic antigen.

Isobaric tags enable multiplexed quantitative analysis of many biological samples in a single LC-MS/MS experiment. As a cost-effective alternative to expensive commercial isobaric tagging reagents, we developed our own custom N,N-dimethyl leucine 'DiLeu' isobaric tags for quantitative proteomics. Here, we present a new generation of DiLeu tags that achieves 21-plex quantification in high-resolution HCD MS/MS spectra via distinct reporter ions that differ in mass from each other by a minimum of 3 mDa. The 21-plex set retains the compact tag structure and existing isotopologues of the 12-plex set but includes nine new reporter variants formulated with unique configurations of 13 C, 15 N, and 2 H stable isotopes, each synthesized in-house via a stepwise N-monomethylation synthesis strategy using readily available reagents. Thus, multiplexing capacity is expanded significantly while preserving the performance and low cost of the previous implementation. We show that 21-plex DiLeu tags generate strong reporter ions following HCD fragmentation of labeled peptides acquired on Orbitrap platforms at a minimum of 60,000 resolving power (at 400 m/z), and we demonstrate accurate 21-plex quantification of labeled K562 human cell line protein digests via single-shot nanoLC-MS/MS analysis on a Q Exactive HF system.

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.

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.

Glycans are highly complex entities with multiple building units and different degrees of branched polymerization. Intensive research efforts have been directed to mass spectrometry (MS)-based qualitative and quantitative glycomic analysis due to the important functions of glycans. Among various strategies, isobaric labeling has become popular because of its higher multiplexing capacity. Over the past few years, several isobaric chemical tags have been developed for quantitative glycomics. However, caveats also exist for these tags, such as relatively low reporter ion yield for aminoxyTMT-labeled complex glycans. To overcome the limitations of existing isobaric chemical tags, we designed a class of novel isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags that can be used to label glycans for quantitative glycomic analysis. The quantitative performance including labeling efficiency, quantification accuracy, and dynamic range of these SUGAR tags has been evaluated, showing promising results. Finally, the 4-plex SUGAR tags have been utilized to investigate N-glycan changes of B-cell acute lymphoblastic leukemia (ALL) pediatric patients before and after chemotherapy.

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 label’s 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 nanoLC-MS2 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 $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.