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Acid/Salt/pH Gradient Improved Resolution and Sensitivity in Proteomics Study Using 2D SCX-RP LC-MS Ming-Zhi Zhu, Na Li, Yi-Tong Wang, Ning Liu, Ming-Quan Guo, Bao-qing Sun, Hua Zhou, Liang Liu, and Jian-Lin Wu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00443 • Publication Date (Web): 28 Jul 2017 Downloaded from http://pubs.acs.org on July 29, 2017

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Journal of Proteome Research

Acid/Salt/pH Gradient Improved Resolution and Sensitivity in Proteomics Study Using 2D SCX-RP LC-MS Ming-Zhi Zhu,†,§ Na Li,† Yi-Tong Wang,† Ning Liu,‡ Ming-Quan Guo,§ Baoqing Sun,‖ Hua Zhou,† Liang Liu,†,* Jian-Lin Wu†,* †

State Key Laboratory for Quality Research of Chinese Medicines, Macau

University of Science and Technology, Macao, China ‡

§

Central Laboratory, Second Hospital of Jilin University, Changchun, China Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture,

Wuhan Botanical Garden, Chinese Academy of Sciences, Sino-Africa Joint Research Center, Chinese Academy of Sciences, Wuhan, China ‖

State Key Laboratory of Respiratory Disease, National Clinical Center for

Respiratory Diseases, Guangzhou Institute of Respiratory Diseases, First Affiliated Hospital, Guangzhou Medical University, Guangzhou, China

1

ACS Paragon Plus Environment


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ABSTRACT: The usage of strong cation exchange (SCX) chromatography in proteomics is limited by its poor resolution, and non-specific hydrophobic interactions with peptides, which lead to peptide overlap across fractions and change of peptide retention, respectively. Due to high salt concentration (up to 1000mM) needed, it also limits to the using of online 2D SCX-RP LC. In the present research, we firstly exploited the chromatographic ability of online 2D SCX-RP LC by combination of acid, salt, and pH gradient, three relatively independent modes of eluting peptides from SCX column. 50% ACN was added to elution buffer for eliminating hydrophobic interactions between SCX matrix and peptides, and the concentration of volatile salt reduced to 50 mM. Acid/salt/pH gradient showed superior resolution and sensitivity, as well as uniform distribution across fractions, consequently leading significant improvements in peptide and protein identification. 112,191 unique peptides and 7,373 proteins were identified by acid/salt/pH fractionation, while 69,870 unique peptides and 4,536 proteins were identified by salt elution, i.e. 62.5% and 60.6% more proteins and unique peptides respectively identified by the former. Fraction overlap was also significantly minimized by acid/salt/pH approach. Furthermore, acid/salt/pH elution showed more identification for acidic peptides and hydrophilic peptides. KEYWORDS: SCX-RP LC, acid/salt/pH gradient, resolution, sensitivity, overlap

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INTRODUCTION In the strategy of shotgun proteomics, the efficient separation of complex protein digests is critical for the comprehensive identification of proteins.1,2 Increasing attention has been paid to multidimensional liquid chromatography (MDLC) separation due to the high peak capacity and excellent resolving power, thereby providing an optimal delivery of peptides into the mass spectrometer.3,4 MDLC is a popular technique that combines two or more orthogonal separation procedures in a consecutive

manner.

Among

MDLC,

two-dimensional

strong

cation

exchange-reversed phase liquid chromatography (2D SCX-RP LC) still represents the mainstay of bottom-up proteomics at present.5-7 In SCX-RP approach, the peptide mixtures are firstly eluted from an SCX column and then subjected to RP chromatography prior to mass spectrometry (MS) analysis. The SCX-RP system exhibits high detection sensitivity and outstanding separation performance due to the high orthogonality between SCX monolithic and RP separation materials.8 Salt and pH elution are two major modes for fractionating SCX-bound peptides.5,9 Among these two modes, salt steps using non-volatile or volatile salts are the most common mode. In this mode, salt is injected directly from autosampler without additional pumps. Salt steps based SCX-RP approach is thus the most simplified commercial 2D system, which is equated most widely by proteomics laboratories in the world.5 However, even small amounts of non-volatile salts accumulated in ion transfer tube also affect ion-transfer efficiency of MS.10 3



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Non-volatile salts are thus rarely used in online SCX-RP-MS/MS platform. Compared with non-volatile salts, volatile salts are, to some extent, tolerated in MS detection. Even so, direct contact of volatile salts with ion source of MS, especially high concentration of volatile salts, still results in source contamination of MS.11 Furthermore, high concentration of salt easily leads to autosampler clogging and capillary blockage, and causes a strong decrease of signal intensity for subsequent MS detection due to the formation of salt adducts.12,13 High concentration of volatile salt is thus generally considered undesirable, and a time-consuming desalting step is required to eliminate salts contained in SCX elutions.13 The mechanism of salt elution in SCX column is that salt competes to replace the peptides adsorbing to the column surface, while pH elution aims to reduce charge number of peptides.14 Although being involved in electrostatic interactions, these two elution modes are relatively independent, and partly complementary to each other.14 Winnik et al.11 developed a 2D SCX-RP method with continuous pH/salt gradient elution for fractionating tryptic digests of human lung epithelial cell lysates. The chromatographic ability of SCX column was greatly exploited by combined pH and salt gradient. However, this method required more sophisticated online setup than salt steps based method, which is not available in all proteomics laboratories. In addition, the elution process was too complicated to optimize. High concentration of volatile salt and narrow pH range also affected the separation effect of SCX chromatography. So the method developed by Winnik et al.11 needs to be simplified and improved for a 4


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wider application. Besides, SCX chromatography also can be eluted by acid gradient, which may be a complementary with salt and pH elution.15 So acid/salt/pH based SCX elution may improve the peptide separation and identification capabilities. Besides electrostatic interactions, SCX matrix also has non-specific hydrophobic interactions with peptides, which is recognized as a major source for peak broadening and peptide lose.13,16 Increasing organic modifier percentage of elution buffer can weaken hydrophobic interactions and increase peptide recovery. It has been reported that peptide recovery from SCX column increased up to five times by increasing acetonitrile (ACN) proportion of elution buffers from 5% to 25% in offline SCX-RP LC system.12 However, the proportion of ACN in elution buffers is usually less than 10% in online SCX-RP LC system. This is because higher percentage of ACN is not compatible with peptide retention in the second-dimensional RP column, leading the flow-through of peptides.9 High percentage of ACN in SCX elution buffers is thus only used in offline multidimensional system.17,18 A solvent-conversion loop between the first and second dimensions is developed in recent years to moderate the solvent composition of elution buffers in various incompatible MDLC, including HILIC-RP and RP-RP.19,20 We used the solvent-conversion loop in the present research to solve the problem of solvent incompatibility. The present work developed a new SCX fractionation method for online SCX-RP LC-MS/MS. The SCX-bound peptides were fractionated by directly injecting the combined acid, salt and pH solutions (containing 50% ACN) from 5



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autosampler into SCX column, and

then

the eluents

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were

diluted by

solvent-conversion loop prior to second-dimensional RP separation. Compared with traditional salt fractionation, acid/salt/pH fractionation showed superior separation efficiency, low overlap and uniform distribution across fractions, which resulted in a 62.5% and 60.6% increase in protein and peptide identification. Moreover, acid/salt/pH fractionation resulted in a larger portion of hydrophilic and acidic peptides being identified. EXPERIMENTAL SECTION Because of space consideration, experimental methods related to chemicals and reagents, sample preparation, MS and data analysis, peptide and protein identification were provided in supporting information. Online Solvent Adjustment Experiments Sample loop was used in the present study for diluting and conditioning the eluted peptide fraction from SCX column. The performance of the sample loop was evaluated and optimized through a flow injection model experiment.21 A mixture of standard protein digests (serum albumin, ovalbumin, β-casein, α-Chymotrypsinogen, cytochrome c, and aprotinin) was injected into the mixing loop prefilled with solvent A (2% ACN with 0.1% formic acid (FA)) through a syringe pump at 1 μL/min for 5 min. Two loop modes and two controls were set up for comparison (Figure S1). The mixture of standard protein digests was dissolved in 5 μL of 50% ACN with 0.1% FA. 20 uL and 40 uL sample loops, which were labeled as modes I and II respectively, 6


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were used for online solvent mixing testing. The protein digests were then loaded into the peptide trap column (Acclaim PepMap, 2 cm × 75 μm, C18, Thermo Scientific). For positive and negative controls, the mixture of standard protein digests was dissolved in 5 μL of 2% ACN with 0.1% FA and 5 μL of 50% ACN with 0.1% FA respectively, and then directly injected to the peptide trap column. 2D SCX-RP LC Analysis 2D SCX-RP LC-MS/MS experiments were performed on a Dionex Ultimate 3000 nano-flow HPLC (Dionex, Sunnyvale, CA). 20 μg of peptides were injected and loaded

into

a

SCX

column

(PolySULFOETHYL ATM,

100 mm × 0.3 mm,

PolyLC Inc., Columbia, MD, USA). Different salt solutions were stored in autosampler. Step salt elution for SCX sub-fractionation was achieved by passing 5 μL of different salt solutions through autosampler. The elution buffer from SCX column was loaded to a 40 μL mixing loop, which was prefilled with solution A. The elution buffer was then transferred to peptide trap column, and the eluted peptides were desalted with solution A at a flow rate of 5 μL/min. Desalting process lasted 10 min for salt concentration ≤ 100 mM, while 20 min for salt concentration > 100 mM. The desalted peptide fractions were further separated in a C18 analytical column (50 cm × 75 μm, Acclaim PepMap RSLC, Thermo Scientific) at a flow rate of 200 nL/min. Peptides were eluted with a nonlinear 90-min gradient from 95% solution B (0.1% FA in H20) to 50% solution C (0.1% FA in ACN).

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2D SCX-RP LC Using Salt Fractionation For salt fractionation, 5, 10, 25, 50, 100, 250, 500, 1000 mM ammonium acetate were used consecutively. The pH values of all these salt solutions were adjusted to 2.7 by FA. The elution buffer flowing out from SCX column was transferred directly to peptide trap column, rather than loading into a 40 μL mixing loop. 2D SCX-RP LC Using Acid/Salt/pH Fractionation For acid/salt/pH fractionation, the elution buffer containing 0.5% TFA and 50% ACN was used for fraction 1; 1% TFA, 50% ACN for fraction 2; 1% TFA, 10 mM ammonium acetate, 50% ACN for fraction 3; 1% TFA, 20 mM ammonium acetate, 50% ACN for fraction 4; 1% TFA, 50 mM ammonium acetate, 50% ACN for fraction 5; 10 mM malonic acid, 0.1% Fa, 50% ACN, and pH were adjusted to 4.0, 6.0 and 8.0 using NH3·H2O for fractions 6-8, respectively. Data Analysis Peptide overlap rates between any two fractions were calculated using following formula: Overlap rate = (overlapping unique peptides between fractions A and B)/[(unique peptides of fraction A + unique peptides of fraction B - overlapping unique peptides between fractions A and B)].22 Physico-chemical properties included theoretical pI value, GRAVY index, as well as the number of acidic amino acid residues and basic amino acid residues in peptides. Among these characteristics, theoretical

pI

value

was

assessed

by

Expasy

8


Proteomics

Server

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(http://web.expasy.org/compute_pi/) and GRAVY index was calculated by online GRAVY Calculator (http://www.gravy-calculator.de/index.php). Results And Discussion Design of Online Solvent Adjustment System It has been demonstrated that addition of organic solvent to elution buffer can weaken hydrophobic interaction between peptides and SCX matrix.16 However, the addition of organic solvent is limited by the miscibility with high concentration of salt. High concentration of organic solvent is also incompatible with peptide retention in the second-dimensional trap column. Thus the percentage of ACN in SCX elution buffer is usually less than 10% in online SCX-RP LC system.9 Xu et al.9 demonstrated that elution buffer containing 50% ACN exhibited highest separation efficiency and largest number of identified peptides for SCX chromatography. We solved solvent incompatibility by placing a sample loop behind SCX column (Figure 1), and 50% ACN was selected for SCX elution buffer in the present research. The elution buffer of 50% ACN was diluted and conditioned in sample loop, which was prefilled with a low-pH, highly aqueous buffer (2% ACN with 0.1% FA). The decrease in the organic content

of

elution

buffer

facilitated

the

focusing

of

peptides

on

the

second-dimensional trap column.23 Previous studies have showed that the volume of sample loop is an important factor affecting the performance of online solvent mixing.21 Herein, 20 μL and 40 μL sample loops were used to evaluate the performance of solvent conversion by injecting a mixture of standard protein digests 9



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(dissolved in 5 μL of 50% ACN with 0.1% FA) (Figure S1). For positive control, the mixture of standard protein digests was dissolved in 2% ACN with 0.1% FA. For negative control, the mixture of standard protein digests was dissolved in 50% ACN with 0.1% FA. The mixture of standard protein digests was then directly injected to the peptide trap column for positive and negative controls. A total of 82 unique peptides were identified by using the 40 μL sample loop, representing an 82.2% increase relative to 45 unique peptides identified by negative control. No marked differences were found between the 40 μL sample loop and positive control. This result showed that perfect mixing was achieved by the 40 μL sample loop. While the similar number of unique peptides was identified between the 20 μL and 40 μL sample loops (73 vs. 82), the ion intensity of chromatographic peaks increased substantially for the latter, especially for hydrophilic peptides. Therefore, the 40 μL loop was sufficient for effective reconstitution of elution buffer for the second-dimensional RP separation in the present system. Evaluation of Acid/Salt/pH Based SCX-RP Platform for Proteome Analysis Salt gradient and pH gradient are two major elution methods for SCX chromatography.5,9 In the present research, we firstly developed an acid/salt/pH based SCX-RP chromatography. To examine the performance of acid/salt/pH based versus salt based 2D SCX-RP LC platforms in proteomics analysis, we analyzed a trypsin-digested complex sample, which was derived from human hepatocellular carcinoma line HepG2/C3A. As showed in table 1, the performance and effectiveness 10


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of acid/salt/pH fractionation were better than those of salt elution for the comprehensive proteomic analyses using a Bruker maXis Impact Q-TOF mass spectrometer. Compared with salt elution, acid/salt/pH elution resulted in identification of 62.5% (7373 vs. 4536) and 60.6% (112,191 vs. 69,879) more proteins and unique peptides respectively in a single run using the same MS acquisition time and sample amount. Similar trend also emerged in triplicate analyses. A total of 7,534 proteins and 146,552 unique peptides were identified by acid/salt/pH approach, while only 4,542 proteins and 80,717 unique peptides were identified by salt elution in triplicate runs, i.e. 65.9% (7,534 vs. 4,542) and 81.6% (146,552 vs. 80,717) more proteins and unique peptides were identified in the former. Besides, the average protein sequence coverage of acid/salt/pH elution (19.4 peptides/protein) was also higher than that of salt elution (17.7 peptides/protein) by triplicate analyses, demonstrating the high confidence level of the acid/salt/pH approach. The separation of SCX chromatography relies on electrostatic interactions between peptides and stationary phase. Multiple charged tryptic peptides usually carry no more than five charges, in which doubly and triply protonated ions are the major ones exceeding 80% in total.24 The very narrow and uneven charged distribution of peptides limits the resolution and separation efficiency, leading to peptide spillover across different SCX fractions.20 This phenomenon decreases sensitivity of the individual peptide in each fraction greatly. It also wastes the finite duty cycle time of MS in redundant peptides, which leads to the clear decrease of identified unique 11



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peptides.21 Overlap is the biggest problem restricting the SCX-RP separation with extensive fractionation.5,24 SCX-RP methodology should to be improved by minimizing the occurrence of peptides splitting among multiple fractions. In the present study, we exploited the chromatographic ability of SCX column by combination of acid, salt, and pH, three relatively independent modes of eluting peptides. To evaluate the efficiency of acid/salt/pH fractionation, the peptide overlap rates between any two fractions were calculated. Figure 2 showed the heatmaps of the unique peptide overlap. A great degree of overlap was observed at salt elution. In this case not only were the overlap rates of adjacent fractions high (about 32%), but also the overlap rates between some distal fractions (for example, 5 and 25 mM) still remained over 10%. Compared with salt elution, the overlap of acid/salt/pH elution was very exciting. The overlap rates of acid/salt/pH elution between neighboring fractions and distal fractions numbered no more than 8% and 3% respectively, suggesting a mild issue with carryover or poor fractionation. These results clearly illustrated that acid/salt/pH elution provided a tremendous improvement in peptide resolution and separation efficiency. Besides

overlap,

another

major

problem

in

salt

elution

is

the

non-uniform distribution of peptides in each SCX fraction.25 The numbers of unique peptides in each SCX fraction were shown in Figure 3. The distribution of peptides was more even throughout all fractions in acid/salt/pH elution, while more than 70% of total unique peptides focused on the first four fractions in salt elution. The 12


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acid/salt/pH elution was thus demonstrated as a better effective online fractionation approach for SCX chromatography. We were also interested in the characteristic of the peptides in each fraction, and wondered if there were any notable differences between these two approaches. Physico-chemical properties of peptides in each fraction were calculated, including theoretical pI value, GRAVY index, as well as the number of acid amino acids and basic amino acids in peptides. As shown in Figure 4A-B, the theoretical pI values had dependence on the order of the fractions in acid/salt/pH elution. The salt elution showed a similar but less remarkable trend. As we known, arginine (R), lysine (K), and histidine (H) residues in peptide sequence are basic, while aspartic acid (D) and glutamic acid (E) residues are acidic.26 As shown in Figure 4C-D, the peptide basicity in acid/salt/pH elution showed clear dependence on the order of fractions. Compared with acid/salt/pH elution, the distribution patterns of the peptide basicity were different in salt elution. The peptides possessing three basic residues were throughout most of the fractions in salt elution, while these peptides were mainly eluted in the last three fractions in acid/salt/pH elution. These results revealed a superior resolution in acid/salt/pH approach. Differing from basicity, acidity did not show the dependency on the order of the fractions in two platforms (Figure S2). In addition, acid/salt/pH and salt elution did not show clear peptide hydrophobicity correlation across the fractions (Figure S3). However, acid/salt/pH fractionation obtained a tremendous increase of hydrophilic and acidic peptide identification (Figure 5). 13



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CONCLUSION In the present research, acid, salt, and pH steps, three relatively independent modes of eluting peptides from SCX column, were used to exploit the chromatographic ability of online 2D SCX-RP LC. Compared with salt fractionation, the acid/salt/pH elution showed superior resolution and sensitivity, low overlap and uniform distribution across fractions, which resulted in a 62.5% and 60.6% increase in protein and peptide identification. Moreover, fractionation results in a larger portion of hydrophilic and acidic peptides being identified. SUPPORTING INFORMATION: The following files are available free of charge at ACS website https://imsva91-ctp.trendmicro.com:443/wis/clicktime/v1/query?url=http%3a%2f%2f pubs.acs.org&umid=EC55A5ED-53D8-D005-A3B3-8EF5315C2AC7&auth=9f5545a dfb06cb9ec0ff28b5960c2c0b5db54f9d-c32634053807802899dc3916f7d69d53acbb48 fa Supplemental experimental section Figure S1: Schematic representation of online solvent adjustment experiments. Figure S2. Distribution of the number of acidic amino acid residues of unique peptides in each fraction eluted by acid/salt/pH and salt approaches. Figure S3. Distribution of GRAVY index value of unique peptides in each fraction eluted by acid/salt/pH and salt approaches.

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File S1.xlsx. Peptides and proteins identification of acid/salt/pH and salt approaches AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (J.-L.Wu) *E-mail: [email protected] (L.Liu) Notes The authors declare no competing financial interest. FUNDING SOURCES: This work is supported by Macau Science and Technology Development Fund (087/2013/A3); Natural science foundation of China (81673580).

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(19) Zhao, Y.; Szeto, S. S. W.; Kong, R. P. W.; Law, C. H.; Li, G. H.; Quan, Q.; Zhang, Z. J.; Wang, Y. Q.; Chu, I. K. Online Two-Dimensional Porous Graphitic Carbon/Reversed Phase Liquid Chromatography Platform Applied to Shotgun Proteomics and Glycoproteomics. Anal. Chem. 2014, 86, 12172-12179. (20) Zhao, Y.; Law, H. C. H.; Zhang, Z. J.; Lam, H. C.; Quan, Q.; Li, G. H.; Chu, I. K. Online coupling of hydrophilic interaction/strong cation exchange/reversed-phase liquid chromatography with porous graphitic carbon liquid chromatography for simultaneous proteomics and N-glycomics analysis. J. Chromatogr. A 2015, 1415, 57-66. (21) Zhao, Y.; Kong, R. P. W.; Li, G. H.; Lam, M. P. Y.; Law, C. H.; Lee, S. M. Y.; Lam, H. C.; Chu, I. K. Fully automatable two-dimensional hydrophilic interaction liquid chromatography-reversed phase liquid chromatography with online tandem mass spectrometry for shotgun proteomics. J. Sep. Sci. 2012, 35, 1755-1763. (22) Zhang, L. F.; Yao, L.; Zhang, Y.; Xue, T.; Dai, G. C.; Chen, K. Y.; Hu, X. F.; Xu, L. X. Protein pre-fractionation with a mixed-bed ion exchange column in 3D LC-MS/MS proteome analysis. J. Chromatogr. B 2012, 905, 96-104. (23) Kong, R. P. W.; Siu, S. O.; Lee, S. S. M.; Lo, C.; Chu, I. K. Development of online

high-/low-pH

chromatography

for

reversed-phase-reversed-phase shotgun

proteomics:

A

two-dimensional

liquid

reversed-phase-strong

cation

exchange-reversed-phase approach. J. Chromatogr. A 2011, 1218, 3681-3688. (24) Wang, H.; Sun, S. N.; Zhang, Y.; Chen, S.; Liu, P.; Liu, B. An off-line high pH 19



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reversed-phase fractionation and nano-liquid chromatography-mass spectrometry method for global proteomic profiling of cell lines. J. Chromatogr. B 2015, 974, 90-95. (25) Lee, H.; Lee, J. H.; Kim, H.; Kim, S. J.; Bae, J.; Kim, H. K.; Lee, S. W. A fully automated dual-online multifunctional ultrahigh pressure liquid chromatography system for high-throughput proteomics analysis. J. Chromatogr. A 2014, 1329, 83-89. (26) Gilar, M.; Jaworski, A.; McDonald, T. S. Solvent selectivity and strength in reversed-phase liquid chromatography separation of peptides. J. Chromatogr. A 2014, 1337, 140-146.

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Figure captions

Figure 1. Schematic of a 2D SCX-RP LC/MS system. Inset: Illustration of the 5 μL partial-loop injection in a 40 μL sample loop. Figure 2. Heatmaps of unique peptide overlap between fractions in acid/salt/pH elution (A) and salt elution (B). Figure 3. Number of unique peptides in each fraction eluted by acid/salt/pH and salt approaches. Figure 4. Comparison of unique peptides in each fraction eluted by acid/salt/pH and salt approaches. A-B: Distribution of theoretical pI value of unique peptides in each fraction eluted by acid/salt/pH and salt approaches; C-D: Distribution of the number of basic amino acid residues of unique peptides in each fraction eluted by acid/salt/pH and salt approaches. Figure 5. Distribution of theoretical pI value (A) and GRAVY index value (B) identified by acid/salt/pH and salt approaches.

21



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Table 1. Protein and peptide identification using salt and acid/salt/pH based platforms. Protein No.

Peptide No.

(1% FDR)

(1% FDR)

salt 1

4,536

69,879

15.4

salt 2

4,537

70,929

15.6

salt 3

4,540

69,657

15.3

salt by triplicate runs

4,542

80,717

17.8

acid/salt/pH 1

7,373

112,191

15.2

acid/salt/pH 2

7,370

112,492

15.3

acid/salt/pH 3

7,387

113,300

15.3

7,534

146,552

19.5

Sample

acid/salt/pH by triplicate runs

22


Average protein sequence coverage (peptides/protein)

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Graphical Abstract 233x138mm (300 x 300 DPI)



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Figure 1. Schematic of a 2D SCX-RP LC/MS system. Inset: Illustration of the 5 µL partial-loop injection in a 40 µL sample loop. 246x145mm (300 x 300 DPI)


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Figure 2. Heatmaps of unique peptide overlap between fractions in acid/salt/pH elution (A) and salt elution (B). 246x101mm (300 x 300 DPI)



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Figure 3. Number of unique peptides in each fraction eluted by acid/salt/pH and salt approaches. 144x77mm (300 x 300 DPI)


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Figure 4. Comparison of unique peptides in each fraction eluted by acid/salt/pH and salt approaches. A-B: Distribution of theoretical pI value of unique peptides in each fraction eluted by acid/salt/pH and salt approaches; C-D: Distribution of the number of basic amino acid residues of unique peptides in each fraction eluted by acid/salt/pH and salt approaches. 254x190mm (300 x 300 DPI)



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Figure 5. Distribution of theoretical pI value (A) and GRAVY index value (B) identified by acid/salt/pH and salt approaches. 226x90mm (300 x 300 DPI)


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pH Gradient Improved Resolution and ... - ACS Publications

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