University of Groningen Selective Maleylation-Directed Isobaric Peptide Termini Labeling for Accurate Proteome Quantification Tian,

Isobaric peptide termini labeling (IPTL) is an attractive protein quantification method because it provides more accurate and reliable quantification information than traditional isobaric labeling methods (e.g., TMT and iTRAQ) by making use of the entire fragment-ion series instead of only a single reporter ion. The multiplexing capacity of published IPTL implementations is, however, limited to three. Here, we present a selective maleylation-directed isobaric peptide termini labeling (SMD-IPTL) approach for quantitative proteomics of LysC protein digestion. SMD-IPTL extends the multiplexing capacity to 4-plex with the potential for higher levels of multiplexing using commercially available C/N labeled amino acids. SMD-IPTL is achieved in a one-pot reaction in three consecutive steps: (1) selective maleylation at the N-terminus; (2) labeling at the ε-NH2 group of the Cterminal Lys with isotopically labeled acetyl-alanine; (3) thiol Michael addition of an isotopically labeled acetyl-cysteine at the maleylated N-terminus. The isobarically labeled peptides are fragmented into sets of band y-ion clusters upon LC-MS/MS, which convey not only sequence information but also quantitative information for every labeling channel and avoid the issue of ratio distortion observed with reporter-ion-based approaches. We demonstrate the SMD-IPTL approach with a 4-plex labeled sample of bovine serum albumin (BSA) and yeast lysates mixed at different ratios. With the use of SMD-IPTL for labeling and a narrow precursor isolation window of 0.8 Th with an offset of −0.2 Th, accurate ratios were measured across a 10-fold mixing range of BSA in a background of yeast proteome. With the yeast proteins mixed at ratios of 1:5:1:5, BSA was detected at ratios of 0.94:2.46:4.70:9.92 when spiked at 1:2:5:10 ratios with an average standard deviation of peptide ratios of 0.34. P quantification gives information on the relative amounts of a large number of proteins between samples. The existing mass-spectrometry-based proteome-wide quantitative methods can be classified into label-free proteomics and label-based proteomics. Even though advanced dataacquisition schemes and algorithms have been developed for label-free proteomics, the limited throughput and signal variation due to, among others, variable sample loss during workup and changing ionization efficiency between injections argue in favor of multiplexed, label-based proteomics. Multiplexed quantification approaches (e.g., ICAT, SILAC, iTRAQ, TMT, and IPTL) exploit different combinations of heavy and light isotopes to differentially label peptides, which enables simultaneous sample workup and LCMS/MS analysis of multiple samples in a single experiment. The commonly used isotopes are C, N, O, and H, with H being less popular because of the potential risk of altering the peptide retention time. The existing multiplexing strategies can also be classified into two categories, MS1 quantification and MS2 quantification, on the basis of the stage at which peptides are quantified. ForMS1 quantification, also called isotopic quantification, such as SILAC and ICAT, the same peptide from different samples will be labeled with different isotopic tags, which results in the same peptide showing multiple precursor ions at the MS1 level. Relative quantification is achieved by comparing the intensities or peak areas of the precursor ions at the MS1 level. Therefore, any isotopic quantification method will at least double the complexity of the MS1 spectrum, which further aggravates the already challenging issue of a limited sampling capacity of precursor ions for MS/MS fragmentation across a chromatographic peak. In contrast, the MS2 quantification methods use isobarically labeled peptides, so the same peptide originating from different samples will have the same mass. After fragmentation, the isobarically labeled peptide will release a unique reporter ion (TMT and iTRAQ) or peptide fragment ions (IPTL), which can be used to reveal the quantification information. TheMS2 quantificationmethods not only allow for the straightforward quantification of multiple samples in a single MS2 spectrum but also further reduce the required instrument time. The most widely used isobaric quantification methods are those using reporter-ion tags (TMT and iTRAQ) because of the multiplex capacity and well-developed data-processing software. However, reporter-ion-based quantificationmethods suffer from Received: March 10, 2020 Accepted: April 22, 2020 Published: April 22, 2020 Article pubs.acs.org/ac © 2020 American Chemical Society 7836 https://dx.doi.org/10.1021/acs.analchem.0c01059 Anal. Chem. 2020, 92, 7836−7844 This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes. D ow nl oa de d vi a U N IV G R O N IN G E N o n Ju ne 4 , 2 02 0 at 0 7: 54 :3 8 (U T C ). Se e ht tp s: //p ub s. ac s. or g/ sh ar in gg ui de lin es f or o pt io ns o n ho w to le gi tim at el y sh ar e pu bl is he d ar tic le s. ratio distortion, which is particularly serious in complex samples, arising from the cofragmentation of multiple peptides passing the precursor-ion selection window. These peptides release identical reporter ions that are indistinguishable in MS2. To correct for the ratio distortion, several methods have been proposed, such as additional gas-phase purification and MultiNotch MS3. An alternative isobaric method that gives rise to multiple quantification ions per peptide and is therefore less affected by the cofragmentation problem is isobaric peptide termini labeling (IPTL). IPTL was first reported in 2009 by Koehler et al., but the approach is not as extensively used as TMT and iTRAQ, presumably because of the limited multiplexing capacity, especially when avoiding deuterium labeling. The initially reported IPTL method showed relative quantification of two samples of LysC digested proteins, where the peptides were crosswise-modified at the Cand Nterminus with a pair of complementary isotopic tags, resulting in isobarically labeled peptides that were fragmented into production clusters. The peptide and protein ratios can be inferred by comparing the intensities of the individual yand b-series fragment ions. Even in the case of cofragmentation of two or more peptides, fragment ions can usually be correctly attributed. IPTL potentially permits more accurate and reliable quantification with multiple quantification data points per spectrum, for each yand b-ion, and suffers less from cofragmentation. Consequently, a number of optimized methods and applications have been reported in the past 10 years, such as selective succinylation and dimethylation based IPTL (triplexIPTL and triplex-QITL), SILAC or proteolytic O labeling combined with IPTL (IVTAL, G-IVTL, QITL, and diDO-IPTL), and pseudoisobaric dimethyl labeling (pIDL, PITL, SWATH-pseudo-IPTL, and MdFDIA). Most of the methods are for duplex labeling or triplex at most, which means that the multiplexing capacity of IPTL is still far less than that of TMT, which has been extended to 16 labeling channels in a single LC-MS/MS run. Recently, Liu et al. reported the pseudoisobaric dimethyl labeling (mpIDL)method, which increased the multiplex capacity to 6-plex. m-pIDL does not suffer from cofragmentation while relying on a wide isolation window of 10 Th. A potential limitation is the utilization of deuterium in the isotopic tags, which carries the risk of changing the retention time of labeled peptides. To improve the IPTL multiplex capacity with nondeuterium tags, we propose the selective maleylation-directed isobaric peptide termini labeling (SMD-IPTL) method, which is based on selective maleylation at the N-termini of LysC digested peptides. The performance of SMD-IPTL was assessed at the 4plex level by spiking different amounts of bovine serum albumin (BSA) into a yeast proteome background. SMD-IPTL can be extended to the 7-plex level using commercially available Cor N-labeled cysteine and alanine. ■ EXPERIMENTAL SECTION Details of the used chemicals and materials, the synthesis of isotopically labeled acetyl-cysteine and the acetyl-alanine pnitrophenol ester, LC purification, LysC digestion, LC/MS/MS analysis, and database searching and quantification can be found in the Supporting Information. Optimization of Selective Maleylation at the Peptide N-Terminus. Solutions of different pH values (7.0, 6.5, 6.0, 5.5, and 5.0) were prepared with 100 mM sodium acetate and acetic acid. Subsequently, the peptide WLYRAK was dissolved in solutions of different pH values (7.0, 6.5, 6.0, 5.5, and 5.0) to a concentration of 10 μM. Then 4 μg/μL maleic anhydride was freshly prepared in acetonitrile and 2 μL was added to 100 μL of each WLYRAK solution. The reaction tube was shaken at room temperature for 30 min. The reaction was tracked with LC-MS. Maleylation on LysC peptides was further optimized by infusing 50 μg/μL maleic anhydride with a syringe pump at a flow rate of 0.4 μL/min into 25 μg of LysC peptides dissolved in 1 mL of sodium acetate−acetic acid solution at pH 5.5 for 1 h. 2-Plex Labeling of Maleylated WLYRAK. Maleylated peptide solution (100 μL) was dried in a vacuum concentrator, followed by the addition of 100 μL of 50 mM sodium tetraborate, and the pH was adjusted to 9 with 500 mM NaOH. Then 100 mM C1-acetyl-alanine p-nitrophenol ester (C1-Ac-Ala-PNP) containing one C label in the acetyl group or acetyl-alanine-p-nitrophenol ester (Ac-Ala-PNP), which contains no C label, was prepared in dimethyl formamide. Subsequently, 2 μL of p-nitrophenol ester was added to the maleylated peptide solution and incubated for 1 h at room temperature. To ensure complete labeling, 2 μL of p-nitrophenol ester was added again and incubated for 30minmore. Afterward, the pH of acetyl-alanine labeled solutions was