Multiple Isotopic Labels for Quantitative Mass Spectrometry

Abstract

Quantitative mass spectrometry is often performed using isotopically-labeled samples. While the 4-trimethylammoniumbutyryl (TMAB) labels have many advantages over other isotopic tags, only two forms have previously been synthesized (i.e. a heavy form containing 9 deuteriums and a light form without deuterium). In the present report, two additional forms containing 3 and 6 deuteriums have been synthesized and tested. These additional isotopic tags perform identically to the previously reported tags; peptides labeled with the new TMAB reagents co-elute from reverse phase HPLC columns with peptides labeled with the lighter and heavier TMAB reagents. Altogether, these 4 tags allow for multivariate analysis in a single liquid chromatography/mass spectrometry analysis, with each isotopically tagged peptide differing in mass by 3 Da per tag incorporated. The synthetic scheme is described in simple terms so that a biochemist without specific training in organic chemistry can perform the synthesis. The interpretation of tandem mass spectrometry data for the TMAB-labeled peptides is also described in more detail. The additional TMAB isotopic reagents described here, together with the additional description of the synthesis and analysis should allow these labels to be more widely used for proteomics and peptidomics analyses.

Cytosolic Carboxypeptidase 1 Is Involved in Processing α- and β-Tubulin

“…Plates were transfected with CCP1 siRNA (mix of three oligonucleotides 10 -20 nM each, Invitrogen) or control siRNA. After incubation for 72 h at 37°C, cells were collected, washed four times in 30 ml of PBS by centrifugation for 5 min at 800 ϫ g at 4°C, and peptides extracted and labeled as described (31,32). Briefly, extracted peptides were labeled with five different 4-trimethylammoniumbutyryl isotopic labels (31,33).…”

“…After incubation for 72 h at 37°C, cells were collected, washed four times in 30 ml of PBS by centrifugation for 5 min at 800 ϫ g at 4°C, and peptides extracted and labeled as described (31,32). Briefly, extracted peptides were labeled with five different 4-trimethylammoniumbutyryl isotopic labels (31,33). Duplicate samples treated with CCP1 siRNA were labeled with the D3-and D9-4-trimethylammoniumbutyryl, and triplicate control samples were labeled with D0-, D6-, and D12-4-trimethylammoniumbutyryl tags.…”

Similar Intracellular Peptide Profile of TAP1/β2 Microglobulin Double-Knockout Mice and C57BL/6 Wild-Type Mice as Revealed by Peptidomic Analysis

“…Sodium phosphate, dibasic anhydrous (Na 2 HPO 4 ), and sodium phosphate monobasic anhydrous (NaH 2 PO 4 ) were from Amresco. The 4-trimethylammoniumbutyryl (TMAB) stable isotopic labeling reagents containing either three, six, and nine atoms of deuterium (D3, D6, and D9-TMAB) or no deuterium (D0-TMAB) were synthesized as described (29)(30)(31) Mice. TAP1/β2m double double-knockout mice were originally provided by Luc Van Kaer from Vanderbilt University School of Medicine Nashville, USA.…”

Limitations of Mass Spectrometry-Based Peptidomic Approaches

“…Peptides were labeled with TMAB using the Nhydroxysuccinimide (NHS) ester of the TMAB reagents, prepared as described [32]. In brief, the TMAB-NHS esters are produced from the reaction of methyl iodide with gammaamino butyric acid in methanol with potassium bicarbonate as a base.…”

“…The TMAB tags were developed by Fred Regnier for proteomic applications, and because they label free amines the TMAB reagents are well suited for peptidomic studies (Regnier et al [31]). The TMAB tags can be produced in five distinct isotopic forms, allowing for multivariate analysis [32]. Using a combination of heat inactivation of the sample and TMAB labels for quantitative peptidomics, we have performed hundreds of LC/MS runs of samples and MS/MS analyses of peptides [20,22,24,25,28,30,[33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48].…”