Mass Defect-Based Pseudo-Isobaric Dimethyl Labeling for Proteome Quantification

Abstract

Discovering differentially expressed proteins in various biological samples requires proteome quantification methods with accuracy, precision, and wide dynamic range. This study describes a mass defect-based pseudo-isobaric dimethyl labeling (pIDL) method based on the subtle mass defect differences between 12 C/ 13 C and 1 H/ 2 H. Lys-C protein digests were labeled with CD 2 O/ 13 CD 2 O and reduced with NaCNBD 3 /NaCNBH 3 as heavy and light isotopologues, respectively. The fragment ion pairs with mass differences of 5.84 mDa were resolved by high-resolution tandem mass spectrometry (MS/MS) and used for quantification. The pIDL method described here resulted in highly accurate and precise quantification results with approximately 100-fold dynamic range. Furthermore, the pIDL method was extended to 4-plex proteome quantification and applied to the quantitative analysis of proteomes from Hca-P and Hca-F, two mouse hepatocarcinoma ascites syngeneic cell lines with low and high lymph node metastasis rates.M ethods of stable-isotope incorporation with mass spectrometry (MS)-based proteome quantification have advanced rapidly in the past decade. Peptide samples can be differentially tagged with heavy or light isotopes by metabolic labeling 1,2 or chemical labeling. 3,4 The mass differences can be distinguished at either the MS or tandem mass spectrometry (MS/MS) level. Dimethyl labeling, 3 a chemical labeling method, is widely used for proteome quantification at the MS level. Several advantages of this method include quick reaction, high labeling efficiency, low cost, and applicability to different types of samples including tissues, cells, and body fluids. 5 Isobaric tags for relative and absolute quantitation (iTRAQ), 4 an MS/MS-based method, allows the proteome quantification of up to eight samples simultaneously and provides more precise quantification results than the MS level quantification method. 6,7 Although these strategies have been widely used for proteome quantification, their accuracy and dynamic range are limited by the signal-tonoise ratio and the increased MS spectral complexity leads to fewer quantified proteins for MS level-based quantification approaches. 8 In addition, ratio distortion caused by precursor interference is common for MS/MS level-based quantification methods. 9,10 To solve these problems, several innovative methods have been recently developed. Isobaric peptide termini labeling (IPTL) 11−13 is an elegant solution in which the amino groups of the N-termini and C-termini of Lys-C protein digests were crosswise labeled with heavy/light isotope reagents according to the slightly different chemical properties of α-and ε-NH 2 . Fragment ion pairs specific to the labeled peptides were used for peptide/protein quantification. Although IPTL improved the quantification accuracy compared to other reported MS/ MS level methods, the side reactions that are inherent in the multistep labeling can adversely affect quantification accuracy and dynamic range. 12 NeuCode SILAC 14 is another st...

Peptide Dimethylation: Fragmentation Control via Distancing the Dimethylamino Group

“…Although dim-4- to dim-6-YGGFLR share a common fragmentation pathway releasing the derivatizing groups as N,N-dimethyl lactams (Scheme 7), the increased distance between the dimethylamino group and the amide bond formed upon the peptide derivatization leads to a discrete increase in the stability of intact precursors from dim-5- to dim-6-YGGFLR, shown as the positive deviation (Figure 7). This agrees with the observation that the ε-dimethylamino group on the lysine side chain is a passive mass tag (Figure S2e), stable to collisional fragmentation in the gas phase [10, 11]. …”

“…Reductive methylation has been used as both mass-difference tagging for MS quantitation [6-8] and recently isobaric mass tagging for MS/MS quantitation [9-11]. Dimethylation is a relatively simple reaction only requiring a reducing agent and formaldehyde.…”

“…In line with recent advancements in the utilization of ultra-high resolving MS for proteomics, differences in mass defects of carbon, hydrogen, oxygen, and nitrogen isotopes have been exploited [10, 11, 16-19]. The scope of isobaric mass tagging is thus being expanded, opening exciting new opportunities in proteome quantitation.…”

“…Both works use Lys-C for protein digestion so that each resulting peptide carries two derivatizing groups. It is interesting to note that there are more derivatized y ions than b ions available to be used for quantitation [10, 11]; in other words the ε-dimethylamino group on the lysine side chain at the peptide C-terminus is more stable than the α-dimethylamino group at the N-terminus.…”

Protected Amine Labels: A Versatile Molecular Scaffold for Multiplexed Nominal Mass and Sub-Da Isotopologue Quantitative Proteomic Reagents

“…In contrast, the advent of mass spectrometers that operate with high duty cycle and resolving power provides an opportunity to quantify isotopologues that contain the same number of neutrons incorporated into different atoms. This approach has previously been utilized for metabolite identification [19], and demonstrated for quantitative proteomics by use of isotopologue-encoded: amino acid NHS-esters [20,21], TMT reagents [22,23], or small molecule protein biochemistry reagents [9,24]. Similarly, metabolic isotopologue reagents were recently described for use in peptide quantification [25,26].…”