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Design of two-dimensional layered double hydroxide nanosheets embedded with Fe3O4 for highly selective enrichment and isotope labeling of phosphopeptides Dandan Jiang, Xiqian Li, and Qiong Jia ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b03806 • Publication Date (Web): 14 Nov 2018 Downloaded from http://pubs.acs.org on November 18, 2018
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Design of two-dimensional layered double hydroxide nanosheets embedded with Fe3O4 for highly selective enrichment and isotope labeling of phosphopeptides Dandan Jiang, † Xiqian Li, ‡ Qiong Jia *, †, § College of Chemistry, Jilin University, Changchun 130012, China (Corresponding author's email:
[email protected]) China-Japan Hospital of Jilin University, Changchun 130033, China § Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China † ‡
KEYWORDS: Layered double hydroxide, Exfoliation, Magnetic, Enrichment, Phosphopeptide
█ INTRODUCTION As a momentous post translational modification, protein phosphorylation regulates various biological processes comprised of cellular growth, division, differentiation, and signaling.1-3 Abnormal protein phosphorylation is closely associated with some human diseases like Alzheimer and Leukemia.4-6 Therefore, the identification of endogenous phosphopeptides in biological samples is crucial for clinical diagnostic and prognosis of diseases.7-9 The complexity of biological samples as well as the weak ionization efficiency and low level of phosphopeptides make mass spectrometry (MS)-based phosphoproteome a big challenge.10,11 To tackle this issue, an effective enrichment for phosphopeptides is prerequisite before MS analysis. To meet this demand, numerous strategies including metal oxide affinity chromatography (MOAC)5 and immobilized metal ion affinity chromatography (IMAC)12 have been developed, which demonstrate amazing potential in phosphopeptides separation. For MOAC, phosphopeptides are captured by Lewis acid-base reaction between them and metal oxides. For IMAC, the interaction between positively charged metal ions and negatively charged phosphate groups of phosphopeptides contribute to the enrichment efficiency. In IMAC technology, metal ions are grafted onto various substrates such as magnetic nanoparticles, columns, and micropipette tips.12-15 However, the surface areas of the adsorbent materials based on these substrates are always small.16 Lately, 2D materials such as graphene or graphene oxide with high surface area has been increasingly adopted as platforms for immobilizing metal ions, allowing the binding of phosphopeptides.17-20 However, graphene-based IMAC materials still have some drawbacks. First, the process of loading metal ions is complicated and time-consuming; second, the performance of graphene-IMAC hybrid materials depends on the content of the loaded metal ions; third, the bound metal ions tend to be easily lost during sample preparation.17,19 Thereby, a novel 2D material with sufficient adsorption capacity toward phosphopeptides via a simple synthesis method is demanded. Layered double hydroxides (LDHs) are a group of 2D layered materials with a formula of [MIIIxMII1−x(OH)2]x+[An−x/n]·mH2O, where MIII and MII represent trivalent and divalent metallic cations (MIII = Fe, Cr, Al; MII = Zn, Cu, Mg, Ni, Co, Ca).21-23 LDHs have acquired extensive applications in many fields due to the biocompatibility, ion exchange capacity, and facile preparation.24-26 In the last decade, exfoliated LDHs have attracted much attention from various researchers. The exfoliation of LDHs relates to a procedure of
swelling and delaminating bulk-size LDHs to open up the inner surface and yield separated nanosheets.27,28 Compared with bulky LDHs, exfoliated LDH nanosheets possess unique 2D single layer structure, abundant positive charge on the surface, and high surface area, benefitting their applications in separation and enrichment field.29,30 However, design and synthesis of LDHs composites for phosphoproteome research remain undeveloped. We herein built a 2D magnetic composite by selecting Cu/Ga LDH as a phosphate affinity material for phosphopeptides enrichment. Considering the high specificity of Cu2+ and Ga3+ toward phosphopeptides, Cu/Ga LDH was prepared and then exfoliated to form single nanosheets to guarantee adequate surface area.12,22,31 Afterward, Fe3O4 nanoparticles were embedded and spread over the nanosheets to maintain the 2D morphology of Cu/Ga LDH. Fe3O4 with good biocompatibility not only exhibited superparamagnetism to facilitate the magnetic separation, but also improved the positive electricity of the material to enhance the adsorption ability of phosphopeptides.32,33 Thanks to the above advantages, Fe3O4-LDH possessed high enrichment capacity, sensitivity, and selectivity in phosphopeptides enrichment, and allowed isotope labeling for investigating the abnormally-regulated phosphopeptides in leukemia patients sera.
█ RESULTS AND
DISCUSSION
Synthesis and characterization of Cu/Ga LDH. In this work, we firstly synthesized Cu/Ga LDH-NO3 materials with different ratios of Cu2+ to Ga3+, after which the exfoliated Cu/Ga LDH (Cu/Ga LDH-Ise) nanosheets were prepared.28,34 Fouriertransform infrared (FT-IR) spectra of Cu/Ga LDH-NO3 (Cu2+:Ga3+ = 3:1 and 2:1) and corresponding Cu/Ga LDH-Ise nanosheets were shown in Figure S1A. By comparing spectra of Cu/Ga LDH-NO3 (curves a and c) and Cu/Ga LDHs-Ise (curves b and d), it could be concluded that the anion exchange was successfully achieved. The absorption band at 1385 cm−1 obviously decreased since NO3– was substituted by Ise–. The absorption peaks at 1045 and 1146 cm−1 were the asymmetric and symmetric stretching vibrations of S=O, indicating that Cu/Ga LDH-Ise composites were prepared with success.35 X-ray photoelectron spectra (XPS) of Cu/Ga LDH-NO3 (Cu2+:Ga3+ = 3:1 and 2:1) and corresponding Cu/Ga LDH-Ise nanosheets were displayed in Figure S1B. N1s (399.9 eV), O1s (530.3 eV), Cu2p (934.8 eV), and Ga2p (1117.9 eV) peaks
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Scheme 1. (A) Synthetic process for Fe3O4-LDH and (B) enrichment procedure of phosphopeptides with Fe3O4-LDH.
appeared in the spectra of Cu/Ga LDH-NO3 (curves a and c). For Cu/Ga LDH-Ise (curves b and d), C1s (284.8 eV) and S2p (160.2 eV) peaks appeared while N1s signal significantly decreased. These results further illustrated that Cu/Ga LDH-NO3 (Cu2+:Ga3+ = 3:1 and 2:1) and the exfoliated nanosheets were successfully synthesized. The contents of Cu2+ and Ga3+ in the above four products were determined by an inductively coupled plasma optical emission spectrometer. The ratios of Cu2+ to Ga3+ were 3.07:1 in Cu/Ga LDH-NO3 (Cu2+:Ga3+ = 3:1), 1.96:1 in Cu/Ga LDH-NO3 (Cu2+:Ga3+ = 2:1), 3.11:1 in Cu/Ga LDH-Ise (Cu2+:Ga3+ = 3:1), and 2.08:1 in Cu/Ga LDH-Ise (Cu2+:Ga3+ = 2:1).36 The structures of the above four products were analyzed by X-ray diffraction (XRD). As could be seen from Figure S1C, the peaks of Cu/Ga LDH-NO3 at 2θ = 11.7°, 23.5°, 34.6°, 39.3°, 43.9°, and 46.7° were assigned to (003), (006), (009), (015), (107), and (018) diffractions corresponding to the regular layered structure, respectively. The (003) and (006) peaks in Cu/Ga LDH-Ise shifted as the exchange proceeded from NO3– to Ise–.28 Atomic force microscopic (AFM) images were used to characterize Cu/Ga LDH-Ise nanosheets using a tapping mode (Figure S2). Results revealed that the thickness of Cu/Ga LDHIse nanosheets was in range of 2–5 nm.28 The surface area and porosity of the composites were investigated by N2 adsorption-desorption analysis. As shown in Figure S3, all of Cu/Ga LDH-NO3 and Cu/Ga LDH-Ise composites exhibited typical type IV adsorption isotherms with H3-type hysteresis loops (P/P0 > 0.8). The isotherms were characteristic of clay minerals, in which large amounts of N2 was adsorbed on slit-shaped mesopores to form multilayer adsorption.37,38 Such results were further evidenced by pore size distributions (Figure S3 insets) calculated by the Barrett-JoynerHalenda (BJH) mode, indicating that the size of mesopores ranged from 2 to 45 nm. The Brunauer-Emment-Teller (BET) surface areas of Cu/Ga LDH-NO3 (3:1) and Cu/Ga LDH-NO3 (2:1) were 102.8 and 113.9 m2 g−1 while those of Cu/Ga LDH-Ise (3:1) and Cu/Ga LDH-Ise (2:1) increased to 220.8 and 230.4 m2 g−1. The above results demonstrated the enhanced surface area after the exfoliation step with Ise–. Zeta potential measurements were employed to monitor the exfoliation of Cu/Ga LDH-NO3. Zeta potential values of Cu/Ga
LDH-NO3 (Cu2+:Ga3+ = 3:1 and 2:1) were determined to be 4.1 and 5.6 mV, respectively. After NO3– was displaced with Ise–, zeta potential values changed to be 21.8 and 24.5 mV. Such results implied the successful exfoliation of LDH-NO3 to form Cu/Ga LDH-Ise. We further studied the adsorption ability of Cu/Ga LDH-Ise composites. A small molecule, p-nitrophenylphosphate (pNPP), was selected for such a goal since it can be adsorbed by phosphate affinity materials due to the existence of a phosphate group.5 As depicted in Figure S4, the saturated adsorption capacity of pNNP with Cu/Ga LDH-Ise (2:1) was measured to be 47.0 μmol g−1 and that with Cu/Ga LDH-Ise (3:1) was 30.1 μmol g−1. The reason why Cu/Ga LDH-Ise (2:1) enhanced the adsorption ability could be explained by its high proportion of Ga3+ compared with that of Cu/Ga LDH-Ise (3:1).12 Thus, Cu/Ga LDH-Ise (2:1) was chosen to deposit Fe3O4 in the following experiments. Synthesis and characterization of Cu/Ga LDH. The preparation process of Cu/Ga LDH-Ise (2:1) embedded with Fe3O4 nanoparticles (hereafter abbreviated as Fe3O4-LDH) was given in Scheme 1.27 Fe3O4 nanoparticles were dispersed onto 2D Cu/Ga LDH-Ise naonosheets instead of common core-shell protocols. Undoubtedly, such a strategy contributed to maintaining adequate surface area, which benefitted subsequent adsorption performance of phosphopeptides. During the construction of Fe3O4-LDH composites, a crucial factor affecting the formation and morphology of Fe3O4 nanoparticles is the dose of Fe source. Figure 1 displayed SEM images of Fe3O4-LDH1, Fe3O4-LDH2, and Fe3O4-LDH3 with different dosages of Fe source (Fe3O4-LDHn was illustrated in Supporting Information). Upon addition of a small quantity of Fe source, Fe3O4 nanoparticles were hard to be observed (Figure 1A). When introducing more Fe source, Fe3O4 nanoparticles were generated and uniformly distributed onto 2D nanosheets (Figure 1B). When the dosage of Fe source further increased, larger particles appeared, which might be attributed to the product of insolution Fe3+/Fe2+ coprecipitation (Figure 1C).5 Since Fe3O4LDH2 exhibited the most satisfactory morphology, its HR-TEM image was investigated (Figure 1D), further indicating the uniform dispersion of Fe3O4 nanoparticles onto LDH nanosheets.
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Figure 1. SEM images of (A) Fe3O4-LDH1, (B) Fe3O4-LDH2, and (C) Fe3O4-LDH3. (D) HR-TEM image of Fe3O4-LDH2. Moreover, the formation of Fe3O4-LDH composites was confirmed with zeta potential measurements. Results were displayed in Figure S5A. The zeta potential of bare Fe3O4 was determined to be 4.3 mV. After Fe3O4 nanoparticles were embedded onto Cu/Ga LDH-Ise, the overall zeta potential values of Fe3O4-LDH composites shifted positively and increased stepwise as increasing dosages of Fe source.5 It is generally known that excellent magnetic response characteristics of magnetic nanoparticles are essential for successful separation and enrichment.39 Vibrating sample magnetometer (VSM) was performed to study the magnetic characteristics of Fe3O4 and Fe3O4-LDH composites (Figure S5B). The saturation magnetization values of Fe3O4, Fe3O4-LDH1, Fe3O4-LDH2, and Fe3O4-LDH3 were 72.43, 32.41, 49.92, and 59.86 emu g−1, respectively. The results indicated that the above products all exhibited a superparamagnetic character. As a representative, Fe3O4-LDH2 could quickly gather with a magnet (Figure S5B inset). In Figure 2A, FT-IR spectra of Fe3O4-LDH composites were shown with Fe3O4-LDH2 as a representative. For comparison, FT-IR spectra of Fe3O4 were also indicated. In curve a, the typical peak at 590 cm−1 belonged to Fe−O−Fe stretching vibrations.40 In the spectrum of Fe3O4-LDH2, the weak absorption peaks at 1146 and 1045 cm−1 together with that of Fe3O4 (590 cm−1) appeared, implying that Fe3O4 was successfully embedded onto Cu/Ga LDH-Ise nanosheets.
XPS results of Fe3O4 and Fe3O4-LDH2 were displayed in Figure 2B. In curve a, the spectrum indicated the presence of O1s and Fe2p peaks. For the spectrum of Fe2p (Figure 2B inset), two characteristic peaks at 724.65 and 710.94 eV belonged to Fe 2p1/2 and Fe 2p3/2 of Fe3O4.41 In curve b, C1s (284.8 eV), Cu2p (934.8 eV), and Ga2p (1117.9 eV) peaks appeared except those of O1s and Fe2p, indicating the success synthesis of Fe3O4-LDH2. XRD patterns of Fe3O4 and Fe3O4-LDH2 were shown in Figure 2C. Fe3O4 and Fe3O4-LDH2 exhibited typical diffractions including (220), (311), (400), (422), (511), and (440) reflection planes, which were indicative of the presence of Fe3O4 crystallite.40 In addition, (003), (006), and (015) reflection planes were clearly observed in curve b, which were typical diffractions of LDH. The surface area and pore structure of Fe3O4-LDH2 were investigated by N2 adsorption-desorption analysis. For comparison, N2 isotherm of Fe3O4 was also measured. As shown in Figure S6A, Fe3O4 exhibited a type V N2 adsorption isotherm with N2 hysteresis loops36,42 while Fe3O4-LDH2 showed a typical type IV adsorption isotherm.38 The pore size distributions of Fe3O4 and Fe3O4-LDH2 were shown in Figure S6A inset which were calculated by the BJH method. The BET surface areas of Fe3O4 and Fe3O4-LDH2 were determined to be 75.0 and 240.3 m2 g−1, respectively. The large surface area of Fe3O4-LDH2 could undoubtedly endow the prepared material with considerable adsorption capacity. The thermal stability of Fe3O4 and Fe3O4-LDH2 were elucidated by thermogravimetric analysis/derivative thermogravimetric (TGA-DTG) determinations. It could be seen from Figure S6B that the weight loss in the whole heating process was slight for Fe3O4 (curve a). The profile of Fe3O4LDH2 exhibited three major regions in the ranges of 25–270, 270–410, and 410–800 °C. The first weight loss was due to the release of water. The weight loss in the second step could be attributed to the removal of nitrate ions and hydroxide ions. Afterwards, the weight declined and then remained essentially unchanged until 800 °C, demonstrating the stable property of Fe3O4-LDH2.34 The intrinsic phosphate adsorption ability of Fe3O4-LDH2 toward pNPP was depicted in Figure 3. For comparison, the adsorption abilities of pNPP with Fe3O4, Fe3O4-LDH1, and Fe3O4LDH3 were also demonstrated. The saturated loading capacity of phosphate with Fe3O4-LDH2 was measured to be 71.3 μmol g−1, which is highest among the four materials. Possible explanations are as followings. When Fe3O4 was immobilized onto Cu/Ga LDH-Ise, Ise− was replaced by Fe3O4 with positive electricity, which benefited the adsorption of phosphate with negative electricity. However, too many Fe3O4 nanoparticles might cause the aggregation phenomenon (Figure 1C), which was unfavorable for the adsorption. Thus, Fe3O4-LDH2 was further studied in the following experiments.
Figure 2. (A) FT-IR spectra, (B) XPS patterns, and (C) XRD curves of (a) Fe3O4 and (b) Fe3O4-LDH2.
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Figure 3. Saturated adsorption isotherms for pNPP adsorbed with (a) Fe3O4, (b) Fe3O4-LDH1, (c) Fe3O4-LDH2, and (d) Fe3O4-LDH3. Method performance of Fe3O4-LDH2 for phosphopeptides enrichment. β-casein tryptic digests were selected as model samples to estimate the enrichment efficiency of phosphopeptides. To achieve high enrichment efficiency, we firstly investigated the concentrations of loading buffers and eluents. To investigate the effects of TFA concentration on the MS intensity, we employed 50% ACN+(1%−5%) TFA (v/v) as loading buffers. It could be observed in Figure S7 that the highest enrichment efficiency could be achieved at 50% ACN+5% TFA. NH3·H2O contents in eluents were optimized in the range from 1% to 10% (Figure S8), revealing that the MS intensity was highest at 5% NH3·H2O. Thus, 50% ACN+5% TFA and 5% NH3·H2O were selected as the optimal loading buffer and eluent for subsequent analysis. Under the optimum conditions, MS peaks achieved without and with Fe3O4-LDH2 enrichment were compared. As demonstrated in Figure S9A, the signals of phosphopeptides could hardly be seen due to the interference of nonphosphopeptides through direct analysis. After the enrichment step, the signals of non-phosphopeptides significantly decreased while those of phosphopeptides could be clearly observed (Figure S9B). These results demonstrated that the present Fe3O4LDH2 material exhibited high enrichment efficiency for phosphopeptides.43 Detailed sequence information was displayed in Table S1. The reason why Fe3O4-LDH2 greatly improved the adsorption ability for phosphopeptides may be interpreted as followings. Firstly, high contents of Cu2+ and Ga3+ contributed to the strong binding affinities for phosphopeptides.12,22,31 Secondly, the adequate surface area offered more opportunities for phosphopeptides to contact with Fe3O4-LDH2. Thirdly, Fe3O4 with positive electricity was propitious to enhance the enrichment efficiency for phosphopeptides. The detection limit of the developed method was evaluated by using β-casein tryptic digests with different amounts, 40, 0.4, 0.02, 0.01 fmol. As shown in Figure S10, 4 phosphopeptides with strong signal intensities could be detected when the concentration was 40 and 0.4 fmol. 2 phosphopeptides peaks existed when the concentration of β-casein tryptic digests was 0.02 fmol. 1 phosphopeptide peak could still be observed at 0.01 fmol, suggesting that the Fe3O4-LDH2-based MALDI MS method had high sensitivity for low level of phosphopeptides. To investigate the enrichment performance of Fe3O4-LDH2 toward phosphopeptides in the presence of non-phosphopeptides, the digests mixture of β-casein and BSA was utilized as a testing sample. It was impossible to identify phosphopeptides signals through direct analysis (Figures 4A, C, and E). However, after enrichment with Fe3O4-LDH2, 7 phosphopeptides peaks could be seen in the mass spectrum of the digests mixture of β-casein and
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BSA with the mass ratio of 1:1000 (Figure 4B). Even when the ratio was further increased to 1:2000 (Figure 4D) and 1:5000 (Figure 4F), the signals of phosphopeptides could still be distinctly identified. These results also pointed to a unique strength of our material in that it allowed for capturing phosphopeptides with high selectivity.44 Considering that practical biosamples contain a large amount of non-phosphopeptides, a more complicated sample was employed to investigate the enrichment performance of Fe3O4LDH2 for phosphopeptides. It could be seen from Figure S11A that no phosphopeptides peaks could be observed in the mass spectrum of digests mixture of β-casein, BSA, and ovalbumin with the mass ratio of 1:5000:5000 through direct analysis. However, 7 phosphopeptides peaks could be observed evidently after enrichment with Fe3O4-LDH2 (Figure S11B), further hinting the highly selective enrichment performance of the prepared material toward phosphopeptides.40 The stability of Fe3O4-LDH2 was investigated with β-casein tryptic digests. As displayed in Figure S12, the signals of phosphopeptides showed little change even after enriching 20 times. Results indicated that the prepared material had good reusability. The S/N ratios of the identified phosphopeptides were displayed in Figure S13. The standard deviations (SD) of the identified phosphopeptides were calculated according to their m/z values (Table S2). The lowest SD was 0.146 at 1561 while the highest SD was 0.440 at 3122, implying that the difference between experiment and theoretics was little.10 We employed a stable isotope labeling method to estimate the recovery of Fe3O4-LDH2 for capture of phosphopeptides.45 Briefly, two samples comprising of the same ration of standard phosphopeptide (LRRApSLGGK) reacted with CH2O or CD2O to result in a mass increase of 28 or 32 Da. After that, the heavy labelled sample was enriched with Fe3O4-LDH2 and the eluent was mixed with the light labelled sample for MS analysis. The signal intensity of the heavy one was divided by the light labelled phosphopeptide to obtain the recovery. It could be seen from Figure 5, the recovery of phosphopeptide was 93.7%, which was competitive among those reported in earlier studies (Table S3).13,17,43-48 In addition, the sensitivity and selectivity of these studies were listed in Table S3, illustrating that Fe3O4-LDH2 was a satisfactory material for capture of phosphopeptides. Biological properties assessment. Human serum and saliva contain many informative peptides such as phosphopeptides generated by pathological tissue, which have attracted growing attention as potential biomarkers of diseases. Matrix effect (ME) of this method was examined since it tends to cause error of analytical results. ME during validation of analytic procedure in biological samples is calculated according to Equation (1):49 ME = (AMS−AM) / AS (1) where AS is the MS peak area of the standard sample (4 pmol βcasein digests) in loading buffer; AM is the area of blank human serum or saliva; and AMS is the area of standard sample in blank sample acquired after the enrichment process. The followings can be described at different ME values: signal enhancement (ME>1.0), no matrix effect (ME=1.0), and signal suppression (ME
