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

Abstract

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.

Figure 1

Overall workflow of quantitative proteomics and amine metabolomics analyses in pancreatic cancer cells using mdDiLeu labeling. Protein and metabolite fractions were separately extracted from PANC1 cells, prepared, and labeled with duplex mdDiLeu reagents. Instrument analyses were performed on the same platform, nanoRPLC–Orbitrap Fusion Lumos Tribid MS.

Figure 2

Optimization of MS parameters for cellular proteomics analysis. (A) NanoRPLC–MS chromatograms of duplex mdDiLeu-labeled peptide and MS spectra under different resolving powers. (B) Numbers of sequenced peak pairs and identified protein groups under different MS resolving powers. (C) Numbers of sequenced peak pairs and identified protein groups under different MS AGC values.

Figure 3

Protein quantification accuracy of mdDiLeu labeling (MS¹-centric quantification) and the comparison with MS/MS reporter ion-based quantification. (A) Box plots represent the measured ratios across all quantified proteins at mixing ratios of 1:1, 1:2, and 1:10. Box denotes 25th and 75th percentiles; line inside the box denotes the median; whiskers denote standard deviation. The dashed lines demarcate the theoretical ratios. (B) The linearity and dynamic range of relative quantification using mdDiLeu (MS¹ quantification) and isobaric DiLeu (MS/MS quantification). Slope =1 and R² = 1 represent perfect linearity and accuracy of relative quantification.

Figure 4

Chromatographic performance of mdDiLeu-labeled cellular metabolites in nanoRPLC–MS. (A) Base peak chromatograms of mdDiLeu-labeled (blue) vs unlabeled (green) metabolites from PANC1 cells. (B) Examples of duplex-labeled cellular amino acids, phenylalanine (Phe), isoleucine (Ile), and leucine (Leu). Isoleucine and leucine can be completely separated after mdDiLeu labeling.

Figure 5

Optimization of MS parameters and quantification accuracy for cellular amine metabolomics analysis. (A) NanoRPLC–MS chromatograms of duplex mdDiLeu-labeled metabolite and MS spectra under different resolving powers. Tryptophan detected from PANC1 cells is used as an example. (B) Numbers of detected total features and quantifiable peak pairs under different MS resolving powers. (C) Numbers of detected total features and quantifiable peak pairs under different MS AGC values. (D) Quantification accuracy of cellular amine metabolites using mdDiLeu labeling. Box plots illustrate the measured ratios across all detected metabolite peak pairs at mixing ratios of 1:1, 1:2, and 1:10, demarcating 25th and 75th percentiles (box), median (line inside the box), standard deviation (whiskers), and the theoretical ratio (dashed line).

Scheme 1. General Structures (A), Synthesis (B), and Activation and Labeling Reactions (C) of Duplex mdDiLeu Reagents^a

^a The light and heavy labels have a mass difference of 20.5 mDa for MS¹-centric quantification.