MS/Ph.D. Qualifying Examination

Department of Chemistry

Subject: Analytical Chemistry

Date: April 24, 2015

Time: (THREE HOURS)

CLOSED BOOK

Instruction: There are THREE sections. You must use

separate BLUE BOOK for each section. Write section

number and question number on the cover of each blue

book.

SECTION-1 Spectroscopy

1. Draw a spectrum of electromagnetic radiation with comparative energy, frequency, and

wavelength. Briefly describe the basic principle of Nuclear Magnetic Resonance (NMR), Infrared

(IR), and Ultraviolet-Visible (UV-Vis) spectroscopic techniques.

2. Briefly describe the basic principle of GC-MS spectrometer. Draw a schematic diagram of GC-MS

instrument including the key compartments.

3. Sketch a comparative molecular orbital diagram of ethylene and 1,3-butadiene. Give an

approximate max value of -* transition of each molecule and briefly explain the difference in your

predicted values.

4. Based on IR, GC-MS, and NMR spectra of unknown compound A (MW = 145.16) shown below,

i) Draw the structure of the compound.

ii) Give the molecular formula of the compound.

iii) Identify the IR peaks of the functional groups in the compound.

iv) Assign the peaks in 1H and

13C NMR spectra to the protons and carbons in the compound.

v) Draw the structure of the fragments responsible for the peaks in the mass spectrum.

5. Based on NMR, IR, and GC-MS spectra of unknown compound B (C6H10O) shown below,

i) Give the degree of unsaturation.

ii) Draw the structure of the fragments responsible for the peaks in the mass spectrum.

iii) Identify the IR peaks of the functional groups in the compound.

iv) Assign the peaks in 1H and

13C NMR spectra to the protons and carbons in the compound.

v) Draw the structure of the compound.

SECTION-2

Electrochemistry

1.) Explain how you would apply potentiometric method in a titration. Describe the experimental

set up, sketch the appearance of data obtained and include an example of the calculation.

2.) You have developed a new metal alloy, which consists of 80% Au and 20% Fe, to be used as

a material for Cl2 gas cylinder. Describe a method to check if your new material would not be

corrosive for its use.

SECTION-3

INSTRUMENTATION (OPTICAL MEASUREMENT AND SEPARATION)

Refer to the article enclosed to answer the following questions:

1. Describe the main function of each major component of the HPLC–triple-quad MS–MS setup

used in the article.

2. What kind of samples can be analyzed by HPLC?

3. How many major components are there in a typical HPLC instrument? What is the function of

each component?

4. How does mass spectrometry work (identify or “weighing” molecules)?

RESEARCH PAPER

Quantitative evaluation of peptide-extraction methods by HPLC–triple-quad MS–MS

Yan Du & Dapeng Wu & Qian Wu & Yafeng Guan

Received: 30 September 2014 /Revised: 25 November 2014 /Accepted: 2 December 2014 /Published online: 27 December 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract In this study, the efficiency of five peptide-extraction methods—acetonitrile (ACN) precipitation, ultra-filtration, C18 solid-phase extraction (SPE), dispersed SPEwith mesoporous carbon CMK-3, and mesoporous silicaMCM-41—was quantitatively investigated. With 28 trypticpeptides as target analytes, these methods were evaluated onthe basis of recovery and reproducibility by using high-performance liquid chromatography–triple-quad tandemmassspectrometry in selected-reaction-monitoring mode. Becauseof the distinct extraction mechanisms of the methods, theirpreferences for extracting peptides of different properties wererevealed to be quite different, usually depending on the pIvalues or hydrophobicity of peptides. When target peptideswere spiked in bovine serum albumin (BSA) solution, theextraction efficiency of all the methods except ACN precipi-tation changed significantly. The binding of BSA with targetpeptides and nonspecific adsorption on adsorbents were be-lieved to be the ways through which BSA affected the extrac-tion behavior. When spiked in plasma, the performance of allfive methods deteriorated substantially, with the number ofpeptides having recoveries exceeding 70 % being 15 for ACNprecipitation, and none for the other methods. Finally, themethods were evaluated in terms of the number of identified

peptides for extraction of endogenous plasma peptides. Onlyultrafiltration and CMK-3 dispersed SPE performed different-ly from the quantitative results with target peptides, and thewider distribution of the properties of endogenous peptideswas believed to be the main reason.

Keywords Method evaluation . Peptide extraction . Plasma .

Triple-quadMS–MS

AbbreviationsDSPE Dispersed solid-phase extractionFA Formic acidMW Molecular weightpI Isoelectric pointQQQ Triple quadrupole

Introduction

Peptide extraction is routine work in many biochemical labo-ratories, because proteolytic digests are usually handled forshot-gun proteomics study [1] and endogenous plasma pep-tides are of great value for biomarker discovery [2–4]. Ad-vances in mass spectrometry (MS) technology and the inte-gration of liquid chromatography (LC) with MS offer extraor-dinary power for peptide identification and quantificationfrom complex biological samples [5, 6].

However, several major challenges still exist in the analysisof endogenous plasma peptides. First, large amounts of intactproteins (e.g. serum albumin at 35–50mgmL−1) and salts willgreatly deteriorate the performance of LC systems or interferewith the signal of peptides in direct MS assays [7]. Second,most valuable peptide biomarkers in plasma are present inconcentrations far below the analytical capabilities of currentMS technology [8], e.g. interleukin 6 at 0–5 pg mL−1 [1].

Electronic supplementary material The online version of this article(doi:10.1007/s00216-014-8389-0) contains supplementary material,which is available to authorized users.

Y. Du :D. Wu (*) :Q. Wu :Y. Guan (*)Key Laboratory of Separation Science for Analytical Chemistry,Department of Instrumentation and Analytical Chemistry, DalianInstitute of Chemical Physics, Chinese Academy of Sciences, 457Zhongshan Road, Dalian 116023, Chinae-mail: dpwu@dicp.ac.cne-mail: guanyafeng@dicp.ac.cn

Y. Du :Q. WuDalian Institute of Chemical Physics, University of the ChineseAcademy of Sciences, Beijing 100039, China

Anal Bioanal Chem (2015) 407:1595–1605DOI 10.1007/s00216-014-8389-0

Third, most peptides are inclined to bind to carrier proteins,for example albumin [2]. Therefore, to enrich peptides anddeplete abundant proteins and salts, peptide extraction is anessential step before LC separation and MS identification.

On the basis of the physicochemical properties of peptides,a variety of extraction methods have been developed. Foracetonitrile (ACN) precipitation, the solubility difference ofpeptides and proteins in organic solvent was exploited toremove high-molecular-weight (MW) protein fraction[9–11]. However, the yield for peptides was low [12], andalbumin at the concentration level of μg mL−1 could still bedetected in the extract of serum [11]. Based on a size-exclusion mechanism [13], centrifugal ultrafiltration was an-other widely used method to remove high-MW proteins[14–18]. Again, the recoveries of peptides were very poor[19]. When the denaturing properties of 20 % (v/v) ACN wereused to dissociate peptides from carrier proteins, more pep-tides could be identified [14].

Solid-phase extraction has been widely used for desaltingproteolytic digests, mainly based on hydrophobic interaction.As well as traditional C18 [14, 20], C8, cationic and anionicexchange, and Oasis HLB adsorbents [19, 21], some newaffinity-extraction materials were also developed, for examplepeptide ligand affinity beads [22]. Recently, ordered mesopo-rous material, MCM-41, was successfully used to selectivelyenrich peptides from human plasma while excluding proteins[23]. Furthermore, ordered mesoporous carbon materialproved to be an outstanding alternative adsorbent for thecapture of endogenous peptides from human plasma [24]. Intotal, 3402 peptides could be identified from only 20 μLhuman plasma by 2D nano-LC–MS–MS analysis.

Because most methods were established using differentsamples and MS techniques, their performance could not becompared directly. Comprehensive comparisons of peptide-extraction methods have been made mainly in terms of thenumber of identified peptides [19, 25–28]. However, to obtaina complete understanding of different extraction methods, itwas necessary to investigate the recoveries of peptides withdifferent properties after treatment by different extractionmethods. So far, there have been few reports on the quantita-tive evaluation of peptide-extraction methods. Potier et al.used selected-reaction-monitoring (SRM) assay coupled withmTRAQ labeling to accurately assess the extraction recover-ies of target peptides from ACN precipitation and ultrafiltra-tion [29]. However, the target peptides were limited to reflectthe extraction efficiency of each method for peptides of dif-ferent properties, several novel extraction materials with highefficiencies were not included, and the effect of the matriceson the extraction of target peptides was not covered.

In this study, three widely used laboratory methods (ACNprecipitation, ultrafiltration, and C18 SPE) and two materialsthat were recently reported to have excellent performance inpeptide extraction (mesoporous silica MCM-41 and

mesoporous carbon CMK-3), were assessed. With 28 trypticpeptides as target analytes, these methods were evaluated onthe basis of recoveries and reproducibility for three sampleswith different matrices (standard samples, spiked bovine se-rum albumin (BSA) samples, and spiked plasma samples).The objective was to achieve a systematic evaluation of pres-ent peptide-extraction methods and offer an evaluation meth-od for emerging methods or materials for peptide extraction.The performance of these methods for extraction of endoge-nous peptides in plasma samples was also evaluated.

Experimental

Chemicals and materials

BSA, ovalbumin (chicken), and formic acid (FA) were obtain-ed from Sigma–Aldrich (St. Louis, MO, USA); dithiothreitol,iodoracetamide, and trypsin were purchased from AladdinChemistry Co., Ltd. (Shanghai, China); human plasma fromhealthy volunteers was provided by the Second AffiliatedHospital of Dalian Medical University and stored in aliquotsat −20 °C; centrifugal filters with a molecular-weight cut-offof 10 kDa were purchased from Millipore (Bedford, MA);C18 SPE cartridges were homemade by packing 50 mg C18beads (50 μm; Merck, Darmstadt, Germany) in 3 mL col-umns; mesoporous materials of MCM-41 (pore size: 33 Å,BET surface area: 921 m2 g−1) and CMK-3 (pore size: 34 Å,BET surface area: 1145 m2 g−1) were purchased from XFNano Inc. (Nanjing, China). Wahaha® purified water (pH~6.5; Hangzhou, China) was routinely used in this study.

Instruments and analytical conditions

An Agilent 1200 series high-performance (HP) LC system(Agilent Technologies, USA) equipped with a diode-arraydetector, coupled with an Agilent 6460 triple-quadrupole(QQQ) mass spectrometer with an electrospray-ionizationsource, was used for quantitative analysis. Chromatographicseparation was achieved on a Zorbax SB-C18 column (2.1×50 mm, 1.8 μm) (Agilent Technologies, USA). The mobilephases were 0.05 % (v/v) FA in H2O (A) and 100 % ACN (B).The LC gradient was: from 5 % B to 26 % B over 42 min at aflow of 0.2 mL min−1, followed by a 5 min wash at 80 % Band a 15 min re-equilibration at 5 % B at a flow of0.4 mL min−1, then 5 % B at a flow of 0.2 mL min−1 for3 min. The QQQ was operated in positive-ion mode. Thenebulizer pressure was 20 psi. Drying-gas flow and tempera-ture were 10 L min−1 and 300 °C, respectively. Sheath-gasflow and temperature were 12 L min−1 and 250 °C,respectively.

All shotgun peptidomic assays were performed on a nano-reversed-phase (RP) LC–MS–MS system consisting of an

1596 Y. Du et al.

LTQ-Orbitrap XL mass spectrometer with a nanospray source(Thermo, San Jose, CA). Enriched peptide sample in 0.1 %FA was loaded on a C18 trap column (200 μm i.d. × 4 cmlength) packed with C18 AQ beads (5 μm, 120 Å) and thenseparated by a homemade C18 capillary column (75 μm i.d. ×17 cm length) packed with C18 AQ beads (3 μm, 120 Å). AFinnigan surveyor MS pump (Thermo, San Jose, CA) wasused to deliver the mobile phase. Buffer A was 0.1 % FA inwater, and buffer B was 0.1 % FA in ACN. The gradient wasprogrammed from 5%B to 35%B (v/v) in 83min with a flowof 300 nL min−1. All MS and MS–MS spectra were acquiredin the data-dependent mode with the ten most intense ionsfragmented by collision-induced dissociation (CID). The fullmass scan performed in the Orbitrap analyzer was acquiredfrom m/z 400–2000 (R=60,000 at m/z 400).

Digestion of standard proteins

BSA and ovalbumin were mixed in an equal molar ratio,denatured by 8 mol L−1 urea, and reduced by dithiothreitolat 56 °C for 1 h. After cooling to room temperature, theproteins were alkylated by iodoracetamide in dark conditionsfor 30min, followed by dilution with 50mmol L−1 NH4HCO3

(pH 8.0). Then trypsin was added, with a weight ratio oftrypsin–protein 1:25, and incubated at 37 °C overnight. Final-ly, the tryptic peptides were dried in a vacuum concentratorand stored at −20 °C.

Selection of target peptides and SRM generation

The tryptic digests of BSA and ovalbumin were profiled onthe HPLC triple-quad MS–MS in MS2 scan mode. Peptideswith no fewer than two charged states observed and those witha substantial signal-to-noise ratio constituted the original twolists of target peptides; however, those with methionine wereexcluded because of possible oxidation. Them/z value of eachselected peptide was calculated from the observed ion with thehighest signal-to-noise ratio. Then the two lists of the calcu-lated m/z values were searched using online Mascot (www.matrixscience.com; Matrix Science Ltd., London, U.K.) withthe following parameters: database, SwissProt; enzyme,trypsin; maximum missed cleavages, two; fixedmodification, carbamidomethyl for the cysteine residues;mass tolerance, 0.9 Da. All the matched peptides of the twolists were finally combined to obtain a set of target peptideswith a wide range of properties. Then the MWand isoelectricpoints (pI) of these peptides were consulted on http://www.expasy.org with ProtParam.

For the design of SRM transitions, the observed ion withhighest signal-to-noise ratio of each target peptide was desig-nated as the precursor ion. MS–MS spectra were obtained onthe HPLC triple-quadMS–MS in product-ionmode. Themostintense fragment ions obtained under lower and higher

collision energies were selected as the product ions for qual-ification and quantification, respectively.

Preparation of samples of different matrices

Three samples of different matrices were prepared as follows:

Peptide-standard samples

The lyophilized digest of BSA and ovalbumin was firstreconstituted to a 56.25 pmol μL−1 stock solution, and thenthe standard sample with a final concentration of 1.125 pmolμL−1 was obtained by further dilution with Wahaha water.

Spiked BSA samples

The BSA stock solution inWahaha water with a concentrationof 1.5 nmol μL−1 was spiked with peptide stock solution, andthen diluted to a final BSA concentration of 30 pmol μL−1 andpeptide concentration of 1.125 pmol μL−1.

Spiked plasma samples

Each plasma aliquot was thawed at room temperature andcentrifuged at 7000g for 8 min. The supernatant was trans-ferred and spiked with peptide stock solution, and then dilutedto a final plasma content of 20 % and peptide concentration of1.125 pmol μL−1.

Peptide extraction

Five peptide-extraction methods, ACN precipitation, ultrafil-tration, C18 SPE, DSPE with CMK-3, and MCM-41, werestudied. For quantitative assessment, 500 μL of the abovethree samples was extracted in triplicate. For the shotgunpeptidomic assay, 500 μL 20 % plasma was used.

ACN precipitation

The procedure was similar to that reported in Ref. [19]. Fororganic-solvent precipitation of proteins, ACN was added tosamples in a 2:1 (v/v) ratio. The mixture was incubated atroom temperature for 30 min and then centrifuged at 12,000gfor 5 min, and the supernatant was collected.

Ultrafiltration

Samples were loaded onto filters for centrifugation at 14,000 gfor 20 min. After washing with 200 μLWahaha water twice,all the filtrates were combined. The retentate was collected bycentrifugation at 800 g for 4 min.

Quantitative evaluation of peptide-extraction methods by HPLC–triple-quad MS–MS 1597

C18 SPE

Peptide extraction by C18 SPE was based on methods report-ed elsewhere [21]. Briefly, the homemade cartridges weresequentially washed with methanol, 50 % ACN–0.05 % FA,and 2 % ACN–0.05 % FA. After loading samples, the car-tridges were washed with 2 % ACN–0.05 % FA, and finallypeptides were eluted with 500 μL 50 % ACN–0.05 % FAthree times.

DSPE using mesoporous materials

The procedure was performed as reported elsewhere [23, 24].Two materials, CMK-3 and MCM-41, were first washed withpure water, and then mixed with samples and shaken for30 min. The mixture was then centrifuged at 12,000 g for10 min to isolate the loaded materials. Next, the materialswere washed twice with 500 μLWahaha water. The peptideswere finally eluted with 50 % ACN for MCM-41 and 50 %ACN–0.05 % FA for CMK-3.

For quantitative assessment, the peptide samples extractedby the five methods were lyophilized to dryness and resus-pended in 500 μL pure water, and 5 μL was injected into theHPLC system. For the shotgun peptidomic assay, the lyoph-ilized extracts were resuspended to a fixed volume and 20 μLwas loaded.

Data analysis

For quantitative analysis, the ratios of the peak areas of sam-ples treated by each method to those of the peptide-standardsolution were calculated as the recoveries of target peptides,and the RSD was calculated for evaluation of the reproduc-ibility of each method.

For shotgun peptidomic analysis, the raw files of MS–MS spectra were searched against the UniProt_Humandatabase using Mascot 2.3 (Matrix Science). The param-eters were: no enzyme; precursor-ion mass tolerance:25 ppm; fragment-ion mass tolerance: 0.8 Da; dynamicmodification: Met (+15.9949 Da). Peptides with ion score≥20 and probability <0.01 were counted as being identi-fied, and these peptides were used to assign the identifi-cation of proteins.

Results and discussion

Establishment of the SRM method of target peptides

For the quantitative method used to evaluate the efficien-cy of peptide-extraction methods, selecting a set of targetpeptides with a wide range of properties was vital. The

tryptic digest of one standard protein contained tens tohundreds of peptides [30], which could be readily pre-pared in the lab. In this study, 28 peptides were selectedfrom the tryptic-digest mixture of BSA and ovalbumin,with MW ranging from 630.9 to 3510.6, and pI valuesfrom 4.09 to 8.75 (Table 1).

Two SRM transitions were designed for each peptide,as shown in Table 1. One transition obtained at lowcollision energy was used for identification, and theother obtained at high collision energy was used forquantification with high sensitivity. To investigate po-tential interference of the plasma matrix when usingthese transitions, plasma pretreated with the ACN-precipitation method was assayed, and no peaks weredetected (Electronic Supplementary Material (ESM)Fig. S1). Thus it was reasonable to use these transitionsto quantify target peptides in the study.

The whole LC–QQQ-MS run was divided into eight timesegments. Only peptides with retention time falling in eachtime segment were monitored during that segment (ESMTable S1).

Quantitative evaluation of peptide-extraction methods

As illustrated in Fig. 1, five peptide-extraction methods, ACNprecipitation, ultrafiltration, C18 SPE, and DSPE with CMK-3 and with MCM-41, were studied, with 28 tryptic peptides astarget analytes. Extraction of peptide-standard sample wasfirst conducted to acquire the inherent extraction efficiencyof each method for peptides of different properties, and thenspiked BSA sample was used to reveal the effect of largeproteins on the extraction process. Finally, the extractionefficiency of these methods for spiked plasma sample wascompared.

Method evaluation with peptide-standard sample

Because of their distinct extraction mechanisms, thesemethods had different preferences for extracting peptides ofdifferent properties, which usually depended on the pI valuesor hydrophobicity of peptides.

ACN precipitation

For ACN precipitation, the recoveries of all the peptidesexceeded 80 %, which indicated good solubility of targetpeptides in 67 % ACN. RSDs of all the target peptides wereless than 12 %.

Ultrafiltration

After treatment by ultrafiltration, the recoveries of targetpeptides were strongly dependent on pI, which has

1598 Y. Du et al.

Tab

le1

Asetofpeptides

selected

forquantitativeevaluatio

nof

differentextractionmethods.T

argetpeptid

ecodes,sequences,molecularweight(MW),isoelectricpoint(pI)values,precursor

ions

and

correspondingcharge

states,productions

forqualificationandquantification,optim

umcollision

energies

andiontypes,andproteins

oforigin

areshow

n

Code

Sequence

MW

(Da)

pIPrecursor

ion(m

/z)

Charge

Production

forqualification

(m/z),collision

energy

(eV),

iontype

Production

forquantification

(m/z),collision

energy

(eV),

iontype

Protein:

BSA

BSA20

ATEEQLK

817.1

4.53

818.1

1+517.0,30,y4

147.2,50,y1

BSA11

LCVLHEK

896.9

6.74

449.6

2+625.3,15,y5

109.9,25,immonium

ionH

BSA10

ECCDKPL

LEK

1290.2

4.68

431.1

3+599.2,10,y5

129,25,[K−H

2O]

BSA5

YICDNQDTISSK

1442

4.21

722

2+584,15,y10

248.8,25,a2

BSA7

LVTDLT

K788.1

5.84

395

2+577.1,10,y5

129.2,25,[K−H

2O]

BSA6

GACLLPK

757

8.22

379.5

2+630.2,10,y5

245.9,25,C

L

BSA8

FKDLGEEHFK

1247.8

5.45

417

3+746.1,10,y6

119.9,25,immonium

ionF

BSA16

LKECCDKPL

LEK

1531.2

6.17

511.5

3+646.3,15,y10

129,25,[K−H

2O]

BSA13

AEFV

EVTK

921

4.53

461.5

2+722.3,10,y6

120,25,immonium

ionF

BSA22

EYEATLEECCAK

1501.2

4.09

751.6

2+796.1,22,y6

264.9,25,Y

E

BSA24

ECCHGDLLECADDR

1748.6

4.1

583.7

3+636.1,13,y5

290.1,25,y2

BSA1

HLV

DEPQ

NLIK

1304

5.32

653.1

2+712.4,25,y6

110,25,immonium

ionH

BSA17

KVPQ

VST

PTLV

EVSR

1638.4

8.75

547.1

3+490.1,13,y4

129.1,25,[K−H

2O]

BSA21

LKPD

PNTLCDEFK

1575.4

4.56

788.7

2+667.9,20,y11

128.9,35,[K−H

2O]

BSA26

KQTA

LVELLK

1141.7

8.59

571.6

2+886.2,20,y8

128.8,25,

[K−H

2O]

BSA31

NECFL

SHKDDSP

DLPK

1901.4

4.66

634.8

3+830.0,18,y14

244,25,y2

BSA28

RPC

FSALT

PDETYVPK

1879.2

6.06

627.5

3+948.2,13,y8

243.9,25,y2

BSA18

ECCHGDLLECADDRADLAK

2246.4

4.23

562.7

4+632.2,10,y11

129.2,25,[K−H

2O]

BSA4

SLHTLFG

DELCK

1418.7

5.3

710.1

2+1081.8,25,y9

110,35,immonium

ionH

BSA23

LKPD

PNTLCDEFK

ADEK

2018.4

4.44

673.8

3+783.8,20,y13

129,35,[K−H

2O]

BSA15

QTA

LVELLK

1013.1

6507.6

2+714.5,10,y6

128.9,25,[K−H

2O]

BSA3

SHCIA

EVEKDAIPENLPP

LTADFA

EDKDVCK

3510.6

4.28

703.1

5+853.2,10,y15

341,25,P

EN

Protein:o

valbum

in

Oval3

LYAEER

778.6

4.53

390.6

2+504.1,10,y4

136.1,22,immonium

ionY

Oval7

ISQAVHAAHAEIN

EAGR

1772.4

6591.7

3+859.3,22,y8

109.9,30,immonium

ionH

Oval11

VYLPR

645.8

8.72

646.8

1+385.3,30,y3

135.8,45,immonium

ionY

Oval1

0GLW

EK

630.9

6631.9

1+462.2,18,y3

158.8,35,immonium

ionW

Oval1

2YPILPE

YLQCVK

1521.4

5.99

761.7

2+1036.1,22,y8

136,35,immonium

ionY

Oval8

ELIN

SWVESQ

TNGIIR

1858.4

4.53

620.3

3+573.1,10,y5

158.9,19,immonium

ionW

Quantitative evaluation of peptide-extraction methods by HPLC–triple-quad MS–MS 1599

rarely been reported before. As shown in Fig. 2, 20peptides out of 28 had recoveries below 80 %. Forthe five most acidic peptides (BSA22, BSA24, BSA18,BSA3, and BSA23; pI below 4.5), more than 49 % wasfound in the retentate, compared with less than 10 %for 21 out of the other 23 peptides. Then the ultrafil-tration membrane was washed with ACN–200 mmol L−1

NH4HCO3=1:4 (v/v) to remove the adsorbed peptides.The washing solution contained more than 10 % of thetotal content of 19 peptides, most of which had high pIvalues. It was speculated that electrostatic interactionhad an important function in the process of ultrafiltra-tion. The membrane was negatively charged at pH 7,meaning acidic peptides were repulsed by nanopores[31], and basic peptides were adsorbed on the mem-brane via electrostatic interaction.

C18 SPE

The recoveries of peptides had a close relationship with re-tention time during RPLC. Both extraction on C18 SPEcartridges and separation on the C18 chromatographic columnwere based on hydrophobic interaction between peptides andthe solid phase. For the latter, retention time reflected thehydrophobicity of analytes. Specifically, peptides eluted ear-lier during RPLC were highly hydrophilic and those elutedlater were highly hydrophobic. Then, the retention time duringRPLC was applied to extraction on C18 cartridges to describethe hydrophobicity of peptides. As shown in Fig. 3, therecoveries of eight peptides out of 11 that eluted earlier werebelow 40%, whereas those of 15 peptides out of 17 that elutedlater were above 80 %.

Interestingly, no peptides except BSA20 were identi-fied in the flow-through fraction (Fig. 3), which indi-cated that most peptides in the sample could becompletely extracted by C18 adsorbents. However, with2 % ACN–0.05 % FA used as the washing solution [19,21] more than 60 % of the loaded amount of mosthydrophilic peptides (eight out of 11) was lost at thewashing step. Then, 0.05 % FA and 5 % ACN–0.05 %FA were used to investigate the effect of the washingsolution. As illustrated in Fig. S2 (ESM), the number ofpeptides with a loss of more than 20 % was five when0.05 % FA was used, compared with 12 for 2 % ACN–0.05 % FA and 17 for 5 % ACN–0.05 % FA. Whenusing 0.05 % FA as washing solution (ESM Fig. S3),the recoveries of most target peptides exceeded 60 %,whereas only the two most hydrophilic peptides hadrecoveries below 30 %. This indicated that C18 SPEwas not suitable for very hydrophilic peptides in

Fig. 2 (a) Schematic diagram of the process for extracting peptides byultrafiltration. (b) Distribution diagram of target peptides in the filtrate,retentate, andwashing solution for preparing peptide-standard samples by

ultrafiltration (n=3). Peptides were ranked by pI values in increasingorder. Conditions: MW cut-off: 10 kDa, washing solution: ACN–200 mmol L−1 NH4HCO3=1:4 (v/v)

Fig. 1 Procedure used for quantitative evaluation of peptide-extractionmethods

1600 Y. Du et al.

biological samples. The optimum washing solution of0.05 % FA was used in the following experiment.

CMK-3 DSPE

CMK-3 was reported to have distinct hydrophobicity, andperformed excellently in peptide extraction [24]. As expected,no peptides were detected in the residuary and washing solu-tions. However, the recoveries of only 15 peptides out of 28exceeded 70 %, and six peptides had recoveries below 50 %.RSDs of 25 peptides were below 11 %. No better result wasobtained when stronger eluate (80 % ACN, 0.05 % FA) wasused (data not shown). It was inferred that another, uncertaininteraction mechanism may have arisen between target pep-tides and the material, which led to incomplete elution.

MCM-41 DSPE

Electrostatic interaction was vital in MCM-41 DSPE. Thedistribution of target peptides in the residuary, washing, andelution solutions had a close relationship with the pI values ofpeptides (Fig. 4). Accordingly, these peptides were dividedinto two groups. For the first group, with lower pI values in therange 4.09–6.00 (from BSA22 to BSA15, but with BSA31,ovalb12, and ovalb7 excluded), 14 out of 18 peptides had atotal loss of more than 60 % in the residuary and washingsolutions. This poor extraction result could be attributed to theelectrostatic repulsion between target peptides and materials,because they were all negatively charged under the loadingcondition (pH 6.5). The second group, with higher pI valuesfrom 6.06 to 8.75 (from BSA28 to BSA17, with BSA31,

Fig. 3 (a) Schematic diagram of the process for extracting peptides byC18 SPE. (b) Distribution diagram of target peptides in the flow-throughfraction, washing solution, and eluate for preparing peptide-standard

samples by C18 SPE (n=3). Peptides were ranked by retention timeduring HPLC. Conditions: washing solution: 2 % ACN–0.05 % FA,eluate: 50 % ACN–0.05 % FA

Fig. 4 (a) Schematic diagram of the process for extracting peptides by MCM-41 DSPE. (b) Distribution diagram of target peptides in the residuarysolution, washing solution, and eluate for preparing standard samples by MCM-41 DSPE (n=3). Peptides were ranked by pI values in increasing order

Quantitative evaluation of peptide-extraction methods by HPLC–triple-quad MS–MS 1601

ovalb12, and ovalb7 included), had no loss in the residuaryand washing solutions. This was ascribed to the strong elec-trostatic attraction between the positively charged peptidesand negatively charged materials. However, the recoveries ofnine of these 10 peptides (the exception being ovalb12) werebelow 61 %, which may be caused by incomplete elution.RSDs of 24 peptides were below 20 %.

Method evaluation with spiked BSA sample

Before the extraction efficiency of these methods for spikedplasma sample was evaluated, spiked BSA sample was usu-ally taken as a model to investigate the effect of proteins on theextraction of peptides.

BSA can bind with target peptides, which may lead to lossof peptides in the extraction process. However, for ACNprecipitation, because of the dissociation effect of ACN therecoveries of 27 peptides out of 28 changed by no more than10 % compared with standard samples. In contrast, the loss

was obvious for ultrafiltration; the recoveries of 13 peptidesdecreased by more than 10 % compared with standard sam-ples (ESMFig. S4a). It was found that the changes for samplescontaining 10 % ACN (v/v) became less than 10 % for allpeptides (ESM Fig. S4b), which was also ascribed to thedissociation effect of ACN.

Regarding the three solid-phase extraction methods, BSAcan adsorb on the surface of the adsorbents, which may changethe extraction behavior. The adsorption amounts of BSA onC18, CMK-3, and MCM-41 were determined to be 8.9, 7.2,and 107.5 mg g−1, respectively. The changes of recoveries ofpeptides compared with standard samples were plotted againstpI values in increasing order. As shown in Fig. S5 (ESM), forC18 SPE the recoveries of several acidic peptides, includingBSA22, BSA24, BSA18, BSA3, and BSA23, decreased bymore than 40 %. Similarly, for CMK-3 DSPE the recoveries ofmost peptides changed little except those of three acidic pep-tides, BSA22, BSA24, and BSA18, which decreased by morethan 20 %. For MCM-41 DSPE, the recoveries of the first

Fig. 5 Distribution of therecoveries of target peptides forpreparing spiked plasma samplesby the five peptide-extractionmethods, and comparison of thenumber of peptides with recover-ies above 70 % obtained bytreating standard samples andspiked plasma samples by the fivemethods (right inset)

Fig. 6 Mass distribution ofpeptides identified after enrichingplasma samples by ACNprecipitation, ultrafiltration, C18SPE, DSPE with CMK-3, andMCM-41

1602 Y. Du et al.

group of acidic peptides changed little (within 10 %), whereasrecoveries for six out of 10 peptides of the second group ofbasic peptides increased by more than 10 %.

The effect of the adsorbed protein on the extraction ofpeptides was clearly revealed by extracting peptide-standardsamples with materials preloaded with BSA. As shown inFig. S6 (ESM), the preloading of BSA led to a higher resid-uary amount of peptides with low pI values for all three SPEmethods.

As illustrated in Fig. S7 (ESM), it was inferred that BSA(pI=4.7) [32] adsorbed on the adsorbents would repel acidicpeptides, meaning a lower amount of peptides would beextracted, as observed for C18 SPE and CMK-3 DSPE. ForMCM-41 DSPE, the six basic peptides may associate withadsorbed BSA and be easily recovered during elution, ac-counting for their increased recoveries.

Generally, proteins adsorbed on SPE materials can changethe extraction behavior of the materials through interactionwith peptides, and ACN can disrupt the binding to someextent.

Method evaluation with spiked plasma samples

Because of the presence of proteins with a wide range ofproperties and at higher content in spiked plasma sam-ples, the extraction efficiency of all these methods dete-riorated substantially for spiked plasma samples. Asshown in Fig. 5, the number of peptides with recoveriesmore than 70 % decreased greatly compared with stan-dard samples. This was believed to be caused by theassociation of target peptides with proteins in the sam-ples and the effect of the protein corona forming on thesurface of adsorbents [33].

Among the five methods ACN precipitation performedbest, with recoveries more than 70 % for 15 peptides, incomparison with none for the other four methods (Fig. 5).The extraction efficiencies of C18 SPE and CMK-3 DSPEwere similar, with close numbers of peptides in differentrecovery ranges. Ultrafiltration and MCM-41 DSPE per-formed worst, with the recoveries of most peptides below20 %.

Extraction of endogenous plasma peptides

For the much more complex set of peptides, endogenousplasma peptides, the extraction efficiency of the methodswas assessed in terms of the number of identified peptides.

As shown in Table S2 (ESM), ultrafiltration andACN precipitation performed best, with 871 and 841unique peptides identified, respectively. Among thethree SPE methods, C18 was most efficient andCMK-3 was poorest (peptide sequences presented inESM Table S3). The results of ACN precipitation,

C18 SPE, and MCM-41 DSPE could be readily under-stood from the above quantitative result. However, theperformance of ultrafiltration and CMK-3 DSPE wasquite different.

The significant deviation between the target-peptidequantitation and plasma-peptide extraction by ultrafiltra-tion may be caused by the difference between the set oftarget peptides and endogenous peptides. All the targetpeptides were tryptic peptides, characterized by a basic Cterminus. Because the ultrafiltration membrane used inthis study was negatively charged, most of the targetpeptides had a different extent of adsorption on themembrane, which resulted in poor recoveries for targetpeptides. However, a large portion of endogenous pep-tides were not R/K terminated, so the loss resulting fromadsorption on the membrane may be alleviated.

With CMK-3 DSPE, the distribution of identified peptideshad a strong dependence onMW, with nearly 90% in the low-MW range (less than 2500 Da) (Fig. 6). The number ofidentified peptides in this MW range was similar to that forthe C18-SPE method, which was in accordance with thequantitative result. However, because of the size-exclusioneffect of the mesopores, this method had poor efficiency forpeptides with high MW, which resulted in the small totalnumber of identified peptides. In addition, because of theexclusion of proteins by the mesopores and extremely lowadsorbed amount of proteins on CMK-3 (compared with themesoporous-silica materials), those peptides that bound tolarge plasma proteins could not be extracted efficiently.

Conclusions

Quantitative evaluation of peptide-extraction methods wasconducted in this study. Twenty-eight tryptic peptides ofstandard proteins (e.g. BSA and ovalbumin, used in thisstudy) with a wide MW and pI distribution covered arelatively wide range of types of peptides, which couldalso be prepared easily across labs. The selection of triple-quad MS–MS for peptide quantification guaranteed reli-able and reproducible results.

ACN precipitation performed best of the five methodsinvestigated, especially for spiked plasma samples. Two pos-sible reasons were suggested. First, the selected target peptidesdissolved very well in 67 % ACN, so there was no obviousloss originating from precipitation. Second, the protein–pep-tide binding could be dissociated effectively by ACN, so theloss from co-precipitation with plasma proteins was mini-mized. Thus, organic-solvent precipitation is very worthy offurther study and improvement, although its enrichment abil-ity is relatively poor and the obtained extract is not fullycompatible with subsequent LC–MS analysis.

Quantitative evaluation of peptide-extraction methods by HPLC–triple-quad MS–MS 1603

For proteolytic digests, C18 SPE is most commonly usedfor peptide enrichment and desalting, with many kinds ofcommercial pipette extraction tip available. It was revealedthat when an appropriate washing solution was used, C18 SPEcould achieve very high recoveries for most tryptic peptides,except for several extremely hydrophilic ones. Despite thislimitation, C18 SPE is still strongly recommended for its greatconvenience.

For peptides in complex matrices, it was found thatthe recoveries of target peptides decreased sharply forall five methods. To achieve high recoveries and cover-age, the following points are suggested on the basis ofthis study.

First, each extraction method is mainly based on one spe-cific mechanism. Because of the wide range of physical andchemical properties of peptides, these methods would natural-ly have discrimination for a part of peptides. To achievecomprehensive extraction of endogenous peptides, two ormore extractionmethods of different principles should be usedin parallel.

Second, the adsorption of peptides on the solid-phase-extraction materials should be both strong and reversible.Strong adsorption could be beneficial to high enrichmentfactors for trace peptides, and reversible adsorption wouldlead to thorough elution. Presently, it was found that somepeptides could not be fully recovered fromMCM-41 or CMK-3. The narrow pore size and complex adsorption mechanismcould be the main reasons.

Third, the capability of solid-phase-extraction material toexclude and resist proteins should be enhanced, because pro-teins adsorbed just inside the pore opening greatly impededthe migration of peptides into the pores. In addition, someadsorbed proteins would be co-eluted with peptides from theadsorbents, which would impair the performance of LC sys-tems or interfere with the direct MS signal. Here, the problemwas resolved mainly through hydrophilic modification of theexternal surface.

Fourth, the binding of endogenous peptides to carrierproteins is a great challenge to thorough peptide extrac-tion. The association can greatly reduce the free con-centration of peptides, and even mask some valuablepeptide biomarkers. Although denaturing reagents, forexample organic solvents and urea, could dissociatepeptides from large carrier proteins, these reagents arenot fully compatible with the peptide-extraction process,because electrostatic interaction, hydrophobic interac-tion, and hydrogen bonds have important functions inboth peptide extraction and protein–peptide binding.

Acknowledgments The financial support by the National NatureScience Foundation of P. R. China (grant Nos. 21005080,21275144, 21475131, 91317313, 21235005, 21321064) is grate-fully acknowledged.

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