Quantitative protein analysis using C7-labeled iodoacetanilide and d5-labeled N-ethylmaleimide by nano liquid chromatography/nano- electrospray ionization ion trap mass spectrometry

Sadamu Kurono a,b, Yuka Kaneko a,b, Satomi Niwayama c,⇑

Abstract

We have developed a methodology for quantitative analysis and concurrent identification of proteins by the modification of cysteine residues with a combination of iodoacet anilide (IAA, 1) and 13C7-labeled iodoace tan ilide (13C7-IA A, 2), or N-eth y lmale imide (NEM, 3) and d5-labe l ed N-eth yl maleim ide (d5-NEM, 4), followed by mass spectro metric analysis using nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry (nano LC/nano-ESI-IT -MS). The combinations of these stable isotope-labeled and unlabeled modifiers coupled with LC separation and ESI mass spectrometric analysis allow accurate quantitative analysis and identification of proteins, and therefore are expected to be a useful tool for proteomics research.

Keywords:
Quantitative analysis
Proteomics
Stable isotope labelling
Cysteine modifiers
Electrospray ionization (ESI)

Introduction

The developme nt of methods for both qualitative and quantitative analysis of proteins has been important in modern proteomics due to their potential for a variety of applications. In particular, stable isotope labeling is becoming a powerful tool. A combination of stable isotope-la beled and unlabeled proteome samples resulting from different external stimuli and subsequent mass spectrometric analysis has been successful ly applied to various systems. Some of the most classical work includes metabolic labeling, wherein cells are cultured in stable isotope-enriched or in normal media for subsequent mass spectrometric analysis of the relative abundanc e of interested proteins. 1–11 The drawbacks to these metabolic labeling experiments include the difficulty of applying them to more complex mammalia n systems, relatively long time frames, and inapplicability to human subjects. On the other hand, isotope tagging of proteome samples by chemical reactions is expected to be applicable to a wider range of proteome samples. Accordingly, pioneering work was reported by Aebersold et al., who employed d8-labeled and unlabeled isotope-coded affinity tags (ICATs) for modification of cysteine residues, 12–15 and their various applicatio ns have been reported. 16–27 However, the ICAT method suffers from several fundamental problems including primary isotope effects exemplified by differential liquid chromatograph y (LC) retention times of d-labeled and unlabeled peptide pairs, 14,28 and complicated collision-induced dissociati on (CID) peptide patterns due to partial fragmentation of the ICAT moieties. These problems are most likely derived from the use of large hydrophobic organic molecules, which have decreased solu- bility in aqueous media. The isobaric tags for relative and absolute quantitati on (iTRAQ) are among the most commonl y applied by labeling primary amino groups. 29–33 Additional methods by label- ing other functional groups have also been reported. 34–38 However, as these reagents do not modify more reactive cysteine residues, an additional alkylation process is required.
We have also been synt hesi zing sma ll orga ni c molec ul es that re- act with spec ific ami no acid res idues and thei r corr es pond ing stab le iso tope -lab eled ver sions to be appl ied to quant ita tive prot eom ic analy sis and iden tification of prot eins . Thes emodifi ersin- clud e N-ethy lm alei mi de (NEM, 3) and d5-labe le d N-ethy lm alei mi de (d5-NEM, 4),39–41 N-b-nap ht hylio do aceta mid e (NBN) and d7-labe led N-b-nap ht hylio do acet am ide (d7-NBN ),42 iodo acet anil ide (IAA, 1) and 13C6 or 13C7-lab el ed iodoa cet oanil ide (13C6-IAA or 13C7-IAA, 2),40,4 1,4 3,44 as well as benz oyloxy su ccini mi des (BSI) and d5-labe le d benz oy loxys uc cini mi des (d5-BSI ).45 We have demo nstr ate d that thes e modi fiers can be succ es sful ly appl ied to quant ita tive anal ysi s of comme rc ial prote in sand mor e compl ex prote om esampl es as wel l as to concu rren t ident ificatio n of prote in s in comb in ation wit h 1D or 2D elec tro phor esi s for sepa ra tion and puri ficatio n of prote in s and mat rix -ass ist ed laser des orpti on /ioni za tion mass spect ro metr y (MALDI- MS ) or elec tro spra y ion izat ion mass spe ctrom et ry (ESI-MS).
The 2D electrophore sis method is still among the most mature biochemical methods for separation of proteins and comprehensi ve analysis of protein expression profiles, often allowing direct determination of protein abundanc e and ready detection of posttranslational modifications. However, it also carries certain problems including its inability to display all proteins, especially low abundance proteins, high or low molecular weight proteins, strongly acidic or basic proteins, or membrane proteins. Recently, therefore, various versions of high-performa nce liquid chromatograph y (HPLC) have become increasingly prevalen t methods for separation of proteins. In fact, most of the methods that apply the organic molecule tagging mentioned above, including ICAT and iTRAQ methods, utilize LC rather than electrophore sis for separation of protein mixtures. Therefore, we examined the applicabil ity of our modifiers to an LC system in order to expand the scope of our method. We previously reported that IAA (and 13C6-IAA) and NEM (and d5-NEM) are the most suitable among the cysteine modifiers we had synthesized due to their solubilities when ESI is utilized. 41 Accordingly, we expected that such labeling reagents would serve as even more useful tools for proteomics research.
Here, we describe the use of stable isotope labeling by a combination of cysteine modifiers, IAA (1) and 13C7-IAA (2), and NEM (3) and d5-NEM (4) (Fig. 1), followed by nano liquid chromatograph y/nanoelectro spray ionization ion trap mass spectrometry (nano LC/nano-ESI-I T-MS) for the relative quantitation of commercial proteins. We also demonstrate that nano LC/nano-E SI-IT-MS allows for confident protein identification and accurate quantitative protein analysis comparable to electro- phoresis and MALDI-MS.
We first attempted to identify amino acid sequences of tryptic peptides modified with IAA (1) or NEM (3).46 For such identification, tandem mass spectrum analysis provides more accurate information than peptide mass finger printing and plays a critical role. Figure 2 shows nano LC/nano-ESI-I T-CID-MS/MS spectra of two IAAs (1)- or NEM (3)-modified tryptic peptide from BSA for identification of amino acid sequences . These spectra indicate that the modifiers remained intact during CID. The dehydratio n (–H2O) peaks are commonly observed in MS/MS spectra of peptides in general. Other spectra of the tryptic peptides modified with two 13C7-IAAs (2) or d5-NEM (4) showed a similar cleavage pattern (data not shown). Moreover , most b and y ions were detected. These ions were all singly-cha rged due to the fact that they derived from doubly- or singly-charge d precursor ions. BSA, OVA, and LCA were identified through protein database searches by the observed tryptic peptides modified with 1 or 3 listed in Tables 1 and 2, respectively.
Individual modification with one 1 (or 2) or with one 3 (or 4) increased the molecular weights of peptides by 133 Da (or 140 Da) or by 125 Da (or 130 Da), respectively, showing that these modifications led to the expected mass difference of 7 Da between 1- and 2-modified peptides and 5 Da between 3- and 4-modified peptides for singly-charged ions including one cysteine residue due to the presence of seven 13C atoms and five deuteriums, respectively . We obtained sequence coverages of 1-modified BSA, OVA, and LCA at 72%, 45%, and 41%, respectively, with compara ble sequence coverages observed without 1-modified protein samples. Therefore, modification with 1 or 2 did not adversely affect protein identification. On the other hand, we obtained less sequence coverages from 3-modified BSA, OVA, and LCA than from these proteins modified with 1. The reasons for this observation appear to be that the number of 3-modified and identified peptides in Table 2 was smaller than that of 1-modified and identified ones in Table 1. Moreover , since unreacte d peptides with 3 or 4 were found while they were rarely seen with 1 or 2, the sequence coverages of 3-modified proteins were low.
We next applied the combinations of 1 and 2 or of 3 and 4 to relative protein quantitation. Scheme 2 shows the workflow for the quantitative analysis of proteins. Three different commercial proteins were prepared in solution at around pH 9.0 for the reaction with 1 (or 2) or 7.0 for the reaction with 3 (or 4). Each sample was mixed with a denaturant and reduced with an excess of reducing agents. Then, each sample was divided into four aliquots for reaction with an excess of 1 or 2 in DMSO and with an excess of 3 or 4 in H2O. Next, the proteins modified with light IAA (1) and those modified with heavy IAA (2) were mixed at various molar ratios (10:1, 6:1, 3:1, 1:1, and 1:3 (light/heavy)), digested with trypsin and analyzed by nano LC/nano-ESI-IT-M S. The proteins modified with light NEM (3) and those modified with heavy NEM (4) were also mixed and prepared in the same manner. The relative monoisotopi c peak area ratios of the extracted ion chromatogra ms (EICs) corresponding to 1- and 2-modified tryptic peptides as well as 3-and 4-modified tryptic peptides were used for quantitative protein analysis.
Next, we applied these cysteine modifiers to the quantitative analysis of proteins modified with 1 and 2 or with 3 and 4 mixed at different molar ratios, including 10 to 1, 6 to 1, 3 to 1, 1 to 1, and 1 to 3. The measured ratio of each mixture was calculated for each peptide shown in Tables 1 and 2 for 1- and 3-modified peptides, respectively, and the ratios for all peptides were averaged. The measuremen ts were obtained three times for each mixture. The results are shown in Tables 3 and 4 for 1- or 2-modified proteins and for 3- or 4-modified proteins, respectively.
We found that the measured relative ratios for each mixture were close to the theoretical molar ratios. Additionally, we ana- lyzed a mixture of BSA, OVA, and LCA with each protein present at a 3 to 1 molar ratio of 1- to 2-modified forms. Each protein was identified and the observed ratios of each protein were accordant with the theoretical ratios (Table 3). Figure 5 shows the observed ratios of 1- to 2- and 3- to 4-modified peptides listed in Tables 3 and 4, respectively, plotted against the theoretical ratios for each protein. The graphs revealed a good correlation between the theoretical and observed ratios for all three proteins. The correlation values between the observed and theoretical ratios for 1- or 2-modified BSA, OVA, and LCA proteins were close to 1 (R2 = 0.9991, 0.9961, and 0.9968; and slope = 0.9808, 1.1157, and 1.1617, respectively) . Therefore, the ionization efficiencies of the IAA (1)-modified peptides and 13C7-IAA (2)-modified peptides were the same within experimental error. When using NEM (3) and d5-NEM (4), the correlation values between the observed and theoretical ratios for 3- or 4-modified BSA, OVA, and LCA proteins were as follows: R2 = 0.9997, 0.9711, and 0.9738; and slope = 0.8756, 1.3426, and 0.7652, respectively. These data suggest that our method allows for the quantitative measurem ent of relative protein ratios with high accuracy particularly using IAA (1) and 13C7-IAA (2). The values of R2s and slopes of 3- or 4-modified OVA and LCA indicate slightly greater discrepancies from the theoretical value (1) than those obtained with IAA (1) and 13C7-IAA (2). The reasons may be attributed to the isotope effects caused by the introduction of five deuteriums and somewhat reduced reactivities of NEM (3) or d5-NEM (4) toward proteins compared to IAA (1) or 13C7-IAA (2) as evidenced by the smaller numbers of identified peptide sequences, although these values are still reasonably close to the theoretical values as we had observed in other samples before. 40 Therefore, they may be improved by refining the reaction conditions for OVA and LCA with 3 or 4. From these results, the combinations of IAA (1) and 13C7-IAA (2) as well as NEM (3) and d5-NEM (4) proved their applicability to quantitati ve analysis of proteins using nano LC/nano-ESI-IT- MS.
In summary, we have demonstrate d that the combination of 13 C7-labeled and unlabeled iodoacetanili de (1 and 2) as well as d5-labeled and unlabeled N-ethylmaleimide (3 and 4) can be a C7-IAA (2) as well as NEM (3) or d5-NEM (4) moiety were observed to be 7, 3.5, and 2.3 Da as well as 5, 2.5, and 1.7 Da apart for singly-, doubly-, and triply-charged species, respectively. This mass difference facilitated the identification of the modified peaks in the LC/MS spectra. Furthermore, we did not observe any isotope effects with respect to peptide retention times, peptide fragmentation, or quantitative protein analysis using IAA (1) or 13 C7-IAA (2).
However, the isotope effect for retention time due to deuterium was observed using NEM (3) and d5-NEM (4), although the quantitative analysis was still acceptable with their use. It is generally reported that d-labeling tends to introduce greater and more frequent isotope effects than 13C-labeling.14 Therefore, this isotope effect observed here is in accordance to this general trend when LC/ESI-MS is utilized, although previously we observed no isotope effect with neither combinatio n of 13C7-IAA (2) and IAA (1) nor of d5-NEM (4) and NEM (3) when electrophore sis and MALDI-MS were utilized. 39,40,44 This isotope effect is likely to become more problematic in analyzing more complex proteome samples. Hence it appears that additional factors including solubility and type of the stable isotope must be taken into account in designing modifiers when LC/ESI-MS system is utilized for quantitati ve analysis of proteins. 47
Despite the drawbacks mentioned above, there are certain merits in using nano LC/nano-ESI-I T-MS in this study in compariso n to using electrophoresis and MALDI-MS. The first merit is its ability to enable comprehensi ve proteom e analysis in an overall shorter time. The nano LC method requires 2 days for the entire process starting with reduction and alkylation of proteins and ending with MS analysis, while the 2D electropho resis method requires more than 3 days for the same samples. The second merit is its ability to separate and distinguis h two or more tryptic peptides with the same molecular weight. For example, two tryptic peptides, which were derived from BSA, detected at the same m/z value 815.8, ETYGDMAD C(IAA, 1)C(IAA, 1)EK and DDPHAC(IAA, 1)YSTVFDK (peptide #10 and peptide #11, respectively, listed in Table 1), were distinguishable using nano LC/nano-E SI-IT-MS, but not 2D electropho resis and MALDI-M S. 40 Therefore, both the character- ization and quantification of proteins are possible using IAA (1) and 13C7-IAA (2) as well as NEM (3) and d5-NEM (4) combined with nano N-Ethylmaleimide LC/nano-ESI-MS/MS or 2D electrophoresis and MALDI-MS /MS, but either method has merits and demerits depending on the nature of the projects, and the choice of the methods depends on the purpose of the projects and the nature of the samples. We are trying to apply this method to clinical proteomics, and the results will be reported in due course.

References and notes

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