Analytical Biochemistry 398 (2010) 34–44

Contents lists available at ScienceDirect

Analytical Biochemistry

journal homepage: www.elsevier .com/locate /yabio

Enrichment of serum low-molecular-weight proteins using C18 absorbentunder urea/dithiothreitol denatured environment

Jing Wu a, Yuan An b, Hai Pu a, Yue Shan a, Xiaoqing Ren a,c, Mingrui An a, Qingsong Wang a,Shicheng Wei c,d,*, Jianguo Ji a,c,*

a National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, People’s Republic of Chinab Department of Rheumatology and Immunology, People’s Hospital, Peking University, Beijing 100034, People’s Republic of Chinac Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, People’s Republic of Chinad Department of Oral and Maxillofacial Surgery, School of Stomatology, Peking University, Beijing 100081, People’s Republic of China

a r t i c l e i n f o

Article history:Received 20 August 2009Received in revised form 19 October 2009Accepted 30 October 2009Available online 3 November 2009

Keywords:SerumLow-molecular-weight proteinEnrichmentC18 absorbentProteomics

0003-2697/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ab.2009.10.047

* Corresponding authors. Addresses: DepartmentSurgery, School of Stomatology, Center for BiomeEngineering, Academy for Advanced Inter- disciplinarNo 5, Yiheyuan Road, Haidian Dist. Beijing 100871, PWei), National Laboratory of Protein Engineering anCollege of Life Sciences, Peking University, No 5, YBeijing 100871, People’s Republic of China (J. Ji). Fax: ++86 10 62751526 (J. Ji).

E-mail addresses: sc-wei@pku.edu.cn (S. Wei), jijg@1 Abbreviations used: LMWP, low-molecular-weight p

lar-weight protein; HSA, human serum albumin; IgG, iacetonitrile; DTT, dithiothreitol; SDS–PAGE, sodium dogel electrophoresis; IEF, isoelectric focusing; Chaps, 3-ylammonio]-1-propane sulfonic acid; Na2S4O6, sodiumtic acid; AgNO3, silver nitrate; BSA, bovine serum albumCHCA, a-cyano-4-hydroxycinnamic acid; ICAT, isotopDulbecco’s modified Eagle’s medium; PBS, phosphategyration; LC, liquid chromatography; MS, mass spectrMS/MS, tandem mass spectrometry; 2-DE, two-dimensimmobilized pH gradient; MALDI, matrix-assisted laseTOF, tandem time-of-flight; S/N, signal/noise; TCEP, trFT–ICR, Fourier transform–ion cyclotron resonance; MCTAP-III, connective tissue-activating peptide III; SPE,

a b s t r a c t

Serum low-molecular-weight proteins (LMWPs, molecular weight <30 kDa) are closely related to thebody physiological and pathological situations, whereas many difficulties are encountered when enrich-ing and fractionating them. Using C18 absorbent (100 Å) enrichment and fractionation under urea/dithi-othreitol (DTT) denatured environment followed by 60% acetonitrile (ACN) elution, serum LMWPs couldbe enriched more than 100-fold and were evaluated by sodium dodecyl sulfate–polyacrylamide gel elec-trophoresis (SDS–PAGE), two-dimensional gel electrophoresis (2-DE), and isotope-coded affinity tag(ICAT) labeling quantification. Proteins existing in human serum at low nanograms/milliliter (ng/ml) lev-els, such as myeloid-related proteins (MRPs), could be identified directly from 2-DE coupled with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI–TOF/TOF MS) andLTQ–Orbitrap MS. Sixteen proteins were confidentially identified and quantified using ICAT labeling andliquid chromatography–tandem mass spectrometry (LC–MS/MS). By virtue of its easy operation and highreproducibility to process large quantity complex serum samples, this method has potential uses inenriching LMWPs either in serum or in cell and tissue samples.

� 2009 Elsevier Inc. All rights reserved.

Serum is a body fluid containing thousands of different proteinswith a huge dynamic concentration range where less than 1% be-long to the serum low-molecular-weight proteins (LMWPs,1

ll rights reserved.

of Oral and Maxillofacialdical Materials and Tissuey Studies, peking University,eople’s Republic of China (S.d Plant Genetic Engineering,iheyuan Road, Haidian Dist.,86 10 62753404 (S. Wei), fax:

pku.edu.cn (J. Ji).rotein; HMWP, high-molecu-

mmunoglobulin gamma; ACN,decyl sulfate–polyacrylamide

[(3-cholamidopropyl) dimeth-thiosulfate; TFA, trifluoroace-in; b-ME, b-mercaptoethanol;e-coded affinity tag; DMEM,-buffered saline; RG, radii ofometry; C, carbamidomethyl;ional gel electrophoresis; IPG,r desorption/ionization; TOF/is(2-carboxyethyl)phosphine;RP, myeloid-related protein;

solid phase extraction.

molecular weight <30 kDa) [1]. Serum LMWPs are composed of cyto-kines, peptide hormones, and proteolytic fragments of larger pro-teins that are of great significance for disease pathological courses.Nevertheless, the study of LMWPs is challenging because most ofthem are low-abundance proteins and high-molecular-weight pro-teins (HMWPs) always interfere with their detection. Thus, it isimperative to enrich LMWPs before their analysis [2].

A variety of methods have been developed to enrich LMWPs. Ingeneral, two major strategies have been adopted in these methods.The first strategy is the immunoaffinity depletion method, whichhas been developed to remove major proteins such as human ser-um albumin (HSA) and immunoglobulin gamma (IgG) [3–9]. Theseaffinity methods can effectively deplete their target proteins (>95%of target proteins can be removed), but the remaining abundantHMWPs (e.g., complements, other immunoglobulin types) can stillinterfere with the detection of LMWPs. Furthermore, manyHMWPs act as carriers of LMWPs, including HSA, the carrier ofmany peptide antigens [10]. When target proteins are depleted un-der native conditions by affinity methods, the LMWPs that arebinding to target proteins are likely to be removed at the sametime [9]. The second strategy is the ultrafiltration method, whichremoves HMWPs according to molecular size [11]. Tirumalai andcoworkers [12] employed denatured solvent conditions (i.e.,

Enrichment of LMWPs using C18 absorbent / J. Wu et al. / Anal. Biochem. 398 (2010) 34–44 35

50 mM NH4HCO3 and 20% acetonitrile [ACN]) for disrupting someprotein–protein interactions and releasing the bound LMWPs soas to reduce the loss of low-molecular-weight binding proteins.However, their buffer system was too soft to break most protein–protein interactions and did not show many protein spots and con-sistent recovery of LMWPs. Recently, Jung and coworkers [13]introduced a urea buffer system (7 M urea, 2 M thiourea, 25 mMNH4HCO3, and 20% ACN at pH 8.2) to serum LMWP analysis bythe ultrafiltration method. This buffer system obtains a high andconsistent recovery rate of LMWPs from human plasma. However,their method also could not effectively remove HMWPs, and theexistence of many HMWPs would interfere with the detection ofLMWPs. In addition, centrifugal filter membranes used in ultrafil-tration methods are always at risk of collapse, and it is also difficultto avoid the loss of LMWPs during the process. Besides immunoaf-finity depletion and the ultrafiltration method, Kay and coworkers[14] introduced an ACN precipitation method to enrich serumLMWPs. This ACN depletion method could remove HMWPs(>75 kDa) but could not effectively enrich LMWPs (<30 kDa) be-cause LMWPs with high abundance could also be depleted by theACN precipitation method. For their invalidation in depletingHMWPs and fractionating LMWPs, none of the affinity, ultrafiltra-tion, or ACN depletion strategies could effectively enrich LMWPs.

We found that when using C18 absorbent under denatured envi-ronment, LMWPs could be effectively separated from HMWPsaccording to their molecular dimension. After serum was incubatedwith C18 absorbent (100 Å) under strong urea/dithiothreitol (DTT)denatured conditions and washed with ACN/H2O gradient bufferto remove HMWPs, a solution with a high concentration of LMWPscould be eluted out. Accordingly, we developed an in-tube chro-matographic absorbent method to enrich LMWPs. In addition, theenriching conditions for human serum LMWPs were optimizedand evaluated by sodium dodecyl sulfate–polyacrylamide gel elec-trophoresis (SDS–PAGE) analysis and quantitative analysis. Thismethod was also successfully applied to fractionate proteins in otherbiological samples such as HEK293 cells and mouse liver tissues.

Materials and methods

Chemical reagents

Sephasil C18 absorbents (60, 100, 200, and 300 Å), octyl-Sephar-ose CL-4B absorbent, HiTrap Blue affinity column and HiTrap rPro-tein A column, and pH 3–10 IEF (isoelectric focusing) strips werepurchased from Amersham Biosciences (Uppsala, Sweden), SilicaGel 60 GF254 was purchased from Merck (Darmstadt, Germany).3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfonicacid (Chaps), DTT, and sodium thiosulfate (Na2S4O6) were pur-chased from Sigma (St. Louis, MO, USA). Trifluoroacetic acid (TFA)and ACN were purchased from Fisher Scientific (Hampton, NH,USA). Urea, Coomassie Brilliant Blue R-250, silver nitrate (AgNO3),bovine serum albumin (BSA), sequencing grade-modified trypsin,and standard protein molecular weight marker were purchasedfrom Promega (Madison, WI, USA). b-Mercaptoethanol (b-ME)was purchased from Fluka (Buchs, Switzerland). A DC Protein As-say Kit was purchased from Bio-Rad (Hercules, CA, USA). a-Cya-no-4-hydroxycinnamic acid (CHCA) matrix was purchased fromBruker Daltonics (Leipzig, Germany). An isotope-coded affinitytag (ICAT) kit was purchased from Applied Biosystems (Foster City,CA, USA).

Fig. 1. Experimental procedure using C18 absorbent for LMWP enrichment andseparation.

Biological samples

The whole blood was collected from 30 healthy persons at thePeople’s Hospital (Beijing, China). After the blood clot at 4 �C for

30 min, serum was collected after centrifuging at 1000g for10 min and was stored at �80 �C until analysis. Human HEK293cells were grown in Dulbecco’s modified Eagle’s medium(DMEM)/10% fetal calf serum. After harvesting, the cells werewashed three times with phosphate-buffered saline (PBS) and col-lected for further treatment. Male mice of the C57BL/6 J strain (9–11 weeks old) were purchased from Beijing Laboratory AnimalCenter (Beijing, China). Liver separation and preparation were con-ducted at 4 �C as described previously [15].

Preparation of LMWPs using C18 absorbent

Serum (1 ml) was dissolved in 3 ml of sample buffer (9 M ureaand 30 mg/ml DTT) at 4 �C for 30 min with occasionally ultrasonicvibration. After centrifuging at 12,000g for 5 min, the supernatantwas collected as the sample solution. Then 1 ml of Sephasil C18

absorbent was rinsed using 4 ml of 90% ACN solution and was cen-trifuged at 1000g for 10 s to remove the liquid phase. Then it wasequilibrated by 4 ml of H2O and centrifuged two times. The samplesolution was incubated with the pretreated C18 absorbent at 4 �Cfor 30 min with occasional vibration. Then it was centrifuged tocollect the supernatant as the unbound fraction, and the C18 absor-bent was washed step by step with 4 ml of 5, 10, 20, and 20% ACNsolution. In each step, the C18 absorbent was washed for 5 min andcentrifuged at 1000g for 30 s to collect the supernatant as thewashed fractions. After the washing steps, 4 ml of 60% ACN solu-tion was added. Following an incubation of 15 min, the absorbentwas centrifuged at 1000g for 30 s to collect the supernatant. Thisstep was repeated twice, and the supernatant was collected andcombined as the eluted fraction. Then the absorbent was cleanedby 4 ml of 90% ACN solution three times, and the cleaned fractionswere collected. The absorbent could be used repeatedly for thesame sample. The procedure is shown in Fig. 1. The unbound,washed, eluted, and cleaned fractions from the C18 absorbent treat-ment procedure were analyzed by 15% Tricine–Tris SDS–PAGE asdescribed previously [16,17].

In addition, other biological samples (cell and tissue extracts)were also tested. HEK293 cell proteins (5 mg) and mouse liver pro-

36 Enrichment of LMWPs using C18 absorbent / J. Wu et al. / Anal. Biochem. 398 (2010) 34–44

teins (5 mg) were resolved by 1 ml of sample buffer (9 M urea and30 mg/ml DTT) and treated by C18 absorbent as described above.The unbound and eluted fractions were collected, dried in vacuum,and analyzed by 15% Tricine–Tris SDS–PAGE.

Molecular weight selectivity of C18 absorbent with different pore sizes

A group of serum proteins was treated individually by C18

absorbent with different pore sizes (60, 100, 200, and 300 Å) as de-scribed above. Their eluted fractions were separated by 15% Tri-cine–Tris SDS–PAGE.

Radii of gyration of proteins under denatured conditions

To better understand the molecular weight selectivity of C18

absorbent with different pore sizes, we examined the radii of gyra-tion (RG) of several standard proteins (BSA, IgG, carbonic anhy-drase, and lysozyme) using an ALV/DLS/SLS-5022F laser lightscattering detector (Department of Chemistry, Peking University)as described by Meyer and coworkers [18]. The measure conditionswere set as follows: temperature, 298.18674 K; viscosity, 0.89040;refractive index, 1.33520; laser wavelength, 632.80000; angle,90.00200; counting time, 300 s. The RG values of both native andurea/DTT denatured proteins were measured and analyzed.

Optimized conditions for enriching human serum

The conditions that influence the enriching efficiency were opti-mized. Six factors were examined and are listed in Table 1: absor-bent type, urea concentration, DTT concentration, denaturingtemperature, sample binding time, and sample protein concentra-tion. SDS–PAGE, gel image, and protein assay were combined todetermine the optimized values.

Enrichment of LMWPs binding to HSA and IgG

During C18 absorbent treatment, LMWPs binding to HSA and IgGwere tested. HSA (50 mg) and IgG (50 mg) were separated from hu-man serum by HiTrap Blue affinity column and HiTrap rProtein Acolumn [19] and then dissolved in 4 ml of sample buffer and trea-ted by the C18 absorbent method as described above. Their elutedfractions were collected and dried in vacuum. Aliquots of raw ser-um, serum-eluted fraction, raw HSA, HSA-eluted fraction, raw IgG,and IgG-eluted fraction were analyzed by means of 12.5% Tris–HClSDS–PAGE according to the manufacturer’s instructions (Bio-Rad).

Tricine–Tris SDS–PAGE analysis and LC–LTQ–Orbitrap MS/MS

As an evaluation of the enriching and fractionating ability of C18

absorbent, four independent groups of raw serum samples andtheir unbound, washed, and eluted fractions were determined bythe DC Protein Assay according to the manufacturer’s instructions(Bio-Rad) with BSA as standard, and then the raw serum samplesand their fractions were dried in vacuum. Aliquots of the driedsamples were analyzed by Tricine–Tris SDS–PAGE and stained with

Table 1Factor values chosen for optimizing LMWP enrichment.

Absorbent type Urea concentration(M)

DTT conce(mg/ml)

Tested values Sephasil C18 0 0Silica Gel 60 GF254 2 3Octyl-Sepharose CL-4B 4 10

8 30Replaced b

Coomassie Brilliant Blue R-250. The gels were digitally scanned,and the images were analyzed by Quantity One software (version4.5.1, Bio-Rad). The total volume of HMWP strips (>30 kDa, includ-ing immunoglobulin light chain strip) and that of LMWP strips(<30 kDa, excluding immunoglobulin light chain strip) were calcu-lated and compared.

Gel bands (<30 kDa) of raw serum or treated serum were cut intosmall pieces, destained in 50% ACN/25 mM NH4HCO3, dehydratedwith ACN, and dried. The dried gel pieces were reduced with10 mM DTT/25 mM NH4HCO3 solution and alkylated with 55 mMiodoacetamide/25 mM NH4HCO3 solution. The dried gel pieces wererehydrated with 5 ng/ll trypsin solution (in 25 mM NH4HCO3) andincubated overnight at 37 �C. Peptides were extracted three timeswith a solution containing 5% formic acid/50% ACN. The digestedpeptides dried with vacuum centrifugation were dissolved in 0.2%formic acid and then separated by a nano-LC (liquid chromatogra-phy) system (Micro-Tech Scientific, Vista, CA, USA) that wasequipped with a C18 reverse phase column (150 lm � 150 mm,5 lm, 120 Å). Samples were separated with a 60-min gradient from5 to 35% ACN at a flow rate of 1 ll/min. Mass spectra were recordedon a linear ion trap orbitrap hybrid mass spectrometer (LTQ–Orbi-trap, Thermo Fisher Scientific, Bremen, Germany). Data acquisitionon the LTQ–Orbitrap was operated in the data-dependent mode.The mass spectrometry (MS) raw data were analyzed using Max-Quant25 software (version 1.0.9.3) and then searched with the Mas-cot search engine against the decoy Human IPI database version 3.39as described previously by Lu and coworkers [20]. The main param-eters were set as follows: fixed modification, carbamidomethyl(Cys); variable modification, N-terminal acetylation and methionineoxidation; maximum number of missing cleavages, 1; MS tolerance,± 7 ppm; tandem mass spectrometry (MS/MS) tolerance, ± 0.5 Da.The required false-positive rate at both the protein and peptide lev-els must be lower than 0.01, and the minimum required peptidelength was set to 6 amino acids.

2-DE separation and MALDI–TOF/TOF MS

Two-dimensional gel electrophoresis (2-DE) was also used todisplay the protein profile before and after C18 absorbent treat-ment. Raw serum sample (100 lg) unbound and eluted fractionproteins were solubilized in 250 ll of 2-DE sample buffer (9 Murea, 65 mM DTT, 4% Chaps, and 0.5% pH 3–10 2-DE ampholytes)[21,22]. After centrifugation for 5 min at 12,000g, the soluble pro-teins were loaded onto nonlinear immobilized pH gradient (IPG)strips (13 cm, pH 3–10 nonlinear, GE Healthcare, Piscataway, NJ,USA). IEF was conducted according to the following procedure:30 V � 8 h, 50 V � 4 h, 300 V � 1 h, 1000 V � 1 h, 3000 V � 1 h,5000 V � 1 h, and 8000 V for a total of 65,000 V h. The seconddimension was performed in 15% Tricine–Tris SDS–PAGE gels thatwere then stained by silver [23,24]. The gels were digitallyscanned, and the images were analyzed by ImageMaster software(version 3.0, Pharmacia Bioscience, Uppsala, Sweden) to comparethe detectable LMWP spot numbers before and after treatment.

Matrix-assisted laser desorption/ionization (MALDI) sampleswere prepared according to the method described by Guerreiro

ntration Denaturingtemperature (�C)

Binding time(min)

Protein concentration(mg/ml)

4 1 30Room temperature 3 570 10 0.5

30 0.05y b-ME 120

Enrichment of LMWPs using C18 absorbent / J. Wu et al. / Anal. Biochem. 398 (2010) 34–44 37

and coworkers [25]. Mass spectra were recorded on an UltraflexMALDI–TOF/TOF (tandem time-of-flight) mass spectrometer (Bru-ker Daltonics) under the control of FlexControl 2.2 software (Bru-ker Daltonics). MALDI–TOF spectra were recorded in the positiveion reflector mode in a mass range from 800 to 4000 Da, and theion acceleration voltage was 25 kV. After analysis of the TOF resultsby FlexAnalysis 2.2, the proteins were subjected to TOF–TOF anal-ysis in ‘‘LIFT” mode. Some of the strongest peaks of each TOF spec-trum were selected as precursor ions that were accelerated in TOF1at a voltage of 8 kV and fragmented by lifting the voltage to 19 kV.Both the MALDI–TOF and MS/MS spectra were processed by Flex-Analysis 2.2 and Biotools 2.2 software and automatically searchedagainst the Swiss-Prot database (version 54.0) using Mascot 2.1.0(Matrix Science, London, UK). The main parameters were set as fol-lows: signal/noise (S/N) >3.0; fixed modification, carbamidomethyl(Cys); variable modification, oxidation (Met); maximum number ofmissing cleavages, 1; MS tolerance, ±100 ppm; MS/MS tolerance,±0.7 Da.

Fig. 3. Molecular weight selectivity of C18 absorbent with different pore sizes. Agroup of serum proteins with the same amount were treated separately by 60, 100,200, and 300 Å C18 absorbents. After stepwise washing by ACN solution as describedabove, aliquots of fractions equivalent to 100 ll of raw serum after 60% ACN elutionwere analyzed using SDS–PAGE for comparison. Lane 1: standard protein marker;lane 2: 10 lg of raw serum; lane 3: elute of 60 Å C18 absorbent; lane 4: elute of 100Å C18 absorbent; lane 5: elute of 200 Å C18 absorbent; lane 6: elute of 300 Å C18

absorbent.

Quantification by ICAT labeling and LC–MS/MS

For specific serum proteins, ICAT labeling quantification wasused to study their individual fold change during the treatment.ICAT labeling was performed with a Cleavable ICAT Reagent Kit(Applied Biosystems) according to the manufacturer’s instructions.Here 100 lg of intact serum proteins and of C18 absorbent-treatedproteins were separately labeled by ICAT light/heavy chain re-agents per the user’s manual (Applied Biosystems). In brief, eachsample suspended in 80 ll of denaturing buffer (50 mM Tris and0.1% SDS at pH 8.5) was reduced with 1 mM tris(2-carboxy-ethyl)phosphine (TCEP) and incubated with cleavable light (12C-la-beled) or heavy (13C-labeled) ICAT reagent at 37 �C for 2 h in thedark. Samples were later combined and digested with trypsin for12 h at 37 �C. The mixture of peptides was cleaned up with cation

Fig. 2. Tricine–Tris SDS–PAGE analysis of serum fractions separated by C18

absorbent. Raw serum (1 ml) dissolved in 3 ml of sample buffer (9 M urea and30 mg/ml DTT) was incubated with C18 absorbent. Then the C18 absorbent waswashed step by step with 4 ml of 5%, 10%, 20%, and 20% ACN solution, followed by60% ACN elution and 90% ACN washing. Aliquots of fractions were subjected to 15%Tricine–Tris SDS–PAGE, and the gel was stained with Coomassie Brilliant Blue R-250. Lane 1: 20 lg of unbound proteins; lanes 2, 3, 4, 5, and 7: amounts of proteinsequivalent to 250 ll of raw serum after stepwise ACN solution washing; lane 6:20 lg of 60% ACN elution samples.

exchange cartridges (Applied Biosystems), and the ICAT-labeledpeptides were purified with avidin cartridges (Applied Biosys-tems). After being left at 37 �C for 2 h with TFA to remove the bio-tin portion of ICAT labels, the purified ICAT-labeled peptides weredried by vacuum centrifuge. The ICAT-labeled peptides were dis-solved in 0.2% formic acid and then separated by a nano-LC system(Micro-Tech Scientific) that was equipped with a C18 reverse phasecolumn (150 lm � 150 mm, 5 lm, 120 Å). Samples were separatedwith a 120-min gradient from 5 to 35% ACN at a flow rate of 400 nl/min. Mass spectra were recorded on a 7-Tesla Fourier transform-ion cyclotron resonance (FTICR) mass spectrometer (APEX-QE, Bru-ker Daltonics). Data were acquired in data-dependent mode using

Fig. 4. RG values of different molecular weight proteins in their intact anddenatured forms. The RG values of four proteins (BSA, IgG, carbonic anhydrase,and lysozyme) were measured using an ALV/DLS/SLS-5022F laser light scatteringdetector. The experiment parameters were as follows: temperature, 298.18674 K;viscosity, 0.89040; refractive index, 1.33520; laser wavelength, 632.80000; angle,90.00200; counting time, 300 s.

38 Enrichment of LMWPs using C18 absorbent / J. Wu et al. / Anal. Biochem. 398 (2010) 34–44

ApexControl 1.0 software (Bruker Daltonics). The strongest peak ofeach MS acquisition was selected for the following MS/MS analysis.The MS/MS spectra were processed by DataAnalysis 3.4 software(Bruker Daltonics) with S/N >4.0 and were automatically searchedagainst the Swiss-Prot database (version 54.0) using Mascot 2.1.0(Matrix Science). Mass tolerances for MS and MS/MS were30 ppm and 0.03 Da, respectively, and variable modifications werespecified as ICAT_heavy and ICAT_light. The instrument setting forthe Mascot search was selected as ‘‘ESI-FTICR.” Finally, the ratio ofthe detected peptide pairs was obtained by calculating their ex-tracted ion chromatograms with WarpLC 1.1 and Biotools 3.1 soft-ware (Bruker Daltonics).

Results

Enrichment of LMWPs

After the treatment of different types of biological samples (hu-man serum, HEK293 cells, and mouse liver extracts) using 100 Å

Fig. 5. Tricine–Tris SDS–PAGE analysis of serum-eluted fractions under different conditidifferent conditions listed in Table 1: (A) replacement of Sephasil C18 absorbent by Silicaand 8 M) used in the sample buffer; (C) increasing concentrations of DTT (0, 3, 10, and 3denaturing temperatures (4 �C, room temperature [RT], and 70 �C); (E) different bindconcentrations of proteins (0.05, 0.5, 5, and 30 mg) used to enrich LMWPs. After the treaand the gels were stained with Coomassie Brilliant Blue R-250.

C18 absorbent, most HMWPs above 30 kDa were separated in theunbound and washed fractions, whereas LMWPs below 30 kDawere enriched in the eluted fractions, as shown in Fig. 2 and Sup-plement Fig. 1 (see supplementary material). Especially, LMWPsbelow 14 kDa were clearly shown after the treatment, in contrastto without the treatment.

Molecular weight selectivity of C18 absorbent with different pore sizes

C18 absorbent with different pore sizes showed different molec-ular weight selectivities (Fig. 3). For 60 Å absorbent, the proteinsbelow 14 kDa were effectively enriched. If the pore size was larger,the molecular weight of enriched proteins was higher. When 300 Åabsorbent was used, proteins above 60 kDa (e.g., HSA) could alsobe enriched. These results indicated that protein molecular sizemight be crucial when binding C18 absorbent under denaturedenvironment. As a validation, the RG values of four proteins (hu-man IgG, BSA, carbonic anhydrase, and lysozyme) under naturaland denatured environments were measured and are shown in

ons. Six groups of serum samples were treated by the C18 absorbent method underGel 60 GF254 or octyl-Sepharose CL-4B; (B) increasing concentrations of urea (0, 2, 4,0 mg/ml) used in the sample buffer or after replacing DTT with b-ME; (D) differenting times of LMWPs to C18 absorbent (1, 3, 10, 30, and 120 min); (F) increasingtment, their 60% ACN-eluted fractions were analyzed by 15% Tricine–Tris SDS–PAGE

Fig. 6. SDS–PAGE analysis of serum, HSA, and IgG samples before and after being separated by C18 absorbent. Raw serum (1 ml), HSA (50 mg), and IgG (50 mg) were dissolvedin 3 ml of sample buffer (9 M urea and 30 mg/ml DTT) and treated by C18 absorbent. Aliquots of samples before and after treatment were subjected to 12.5% Tris–HCl SDS–PAGE and stained with Coomassie Brilliant Blue R-250. Lane A1: molecular weight markers; lane A2: 20 lg of raw serum before treatment; lane A3: serum-eluted fractionafter treatment (protein amounts equivalent to 200 ll of raw serum after 60% ACN elution); lane B1: 20 lg of HSA before treatment; lane B2: HSA-eluted fraction aftertreatment (protein amounts equivalent to 50 mg of HSA after 60% ACN elution); lane C1: 10 lg of IgG before treatment; lane C2: IgG-eluted fraction after treatment (proteinamounts equivalent to 50 mg of IgG after 60% ACN elution).

Table 2Enrichment of serum proteins in the raw and eluted fractions.

Group Raw serum Eluted fraction

HMWPs(%)

LMWPs(%)

Proportion toraw serum (%)

HMWPs(%)

LMWPs(%)

1 99.2 0.8 1.0 5.8 94.22 99.2 0.8 0.9 6.2 93.8

Enrichment of LMWPs using C18 absorbent / J. Wu et al. / Anal. Biochem. 398 (2010) 34–44 39

Fig. 4. The curve of relationship between the RG of intact proteinsand molecular weight was a power function (RG = 4.856 �M0.5854,where M is molecular weight [kDa] and R2 = 0.991), whereas theRG of denatured proteins was linearly correlated with their molec-ular weight (RG = 4.091 �M � 18.576, where M is molecularweight [kDa] and R2 = 0.991). These data also corresponded withthe selectivity of C18 absorbent with different pore sizes.

3 99.1 0.9 0.8 3.5 96.54 99.2 0.8 1.0 5.1 94.9Mean 99.2 0.8 0.9 4.9 95.1

Optimized conditions for enriching human serum

Six groups of human serum were treated by C18 absorbent underdifferent conditions. SDS–PAGE (Fig. 5) showed that absorbent type,urea concentration, and DTT concentration were crucial. Replace-ment of Sephasil C18 absorbent by Silica Gel 60 GF254 or octyl-Se-pharose CL-4B and absence or low concentration of urea or DTTwould lead to a nonideal separation. Although 4 M urea and 3 mg/ml DTT were necessary, more urea or DTT made no significant differ-ence and DTT could be replaced by b-ME. The denaturing tempera-ture also influenced the separation effect. Although highdenaturing temperature made more LMWPs detected, it might causemodification or degradation of proteins, so 4 �C was selected as thedenaturing temperature for further serum treatment. When bindingtime was longer than 3 min, no significant difference was displayed,indicating that the binding between LMWPs and C18 absorbent was ashort-term course. Moreover, because the sample protein concen-tration did not affect the separation, this method was compatiblewith a large concentration range of total serum proteins.

Taken as a whole, the optimized conditions for enrichment ofserum LMWPs were as follows: (i) C18 was chosen for absorbenttype; (ii) urea and DTT concentrations were separately above 4 Mand 3 mg/ml, respectively; (iii) binding time was longer than3 min; (iv) denaturing temperature was 4 �C; (v) sample proteinconcentrations could be varied as needed.

Enrichment of LMWPs binding to HSA and IgG

SDS–PAGE analysis was carried out on the serum, HSA, and IgGsamples treated or untreated by C18 absorbent. The loading

amount did not correspond to the equal value, but the results dis-played the actual distribution of proteins before and after treat-ment. It was clearly shown that LMWPs below 30 kDa could besignificantly enriched after 100 Å C18 absorbent treatments(Fig. 6, lane A3) compared with those before treatments (Fig. 6,lane A2). The SDS–PAGE results also showed that some LMWPs,which would bind to HSA or IgG when using the affinity methodfor separation (Fig. 6, lanes B1 and C1), could also be releasedand enriched by the C18 absorbent method (Fig. 6, lanes B2 and C2).

SDS–PAGE analysis of LMWPs

Four independent replicates of raw serum samples and theireluted fractions were determined by protein assay and SDS–PAGEimage analysis. The distribution percentages of HMWPs andLMWPs in each fraction are presented in Table 2. Compared withthe raw serum samples, the mean content of LMWPs (<30 kDa,excluding immunoglobulin light chains) in the elution fractionwas raised from less than 0.9% to more than 95%, an increase ofmore than 100 times. The remaining HMWPs in the eluted fractionwere identified using MALDI–TOF MS/MS and were albumin, a2-antitrypsin, and immunoglobulin light chain. The total content ofthe remaining HMWPs was less than 10%, and they were unlikelyto interfere with the detection of LMWPs. It was also shown thatthe maximum difference of LMWP pattern among four replicateswas 2.7%, which represented a high reproducibility.

Table 3Spots of LMWPs separated by 2-DE before and after enrichment using C18 absorbent.

Group LMWP (MW < 30 kDa) spots

Gel 1 Gel 2 Gel 3 Mean ± SD

Raw serum 128 135 140 134 ± 6Unbound fraction 85 76 80 80 ± 5Eluted fraction 292 321 308 307 ± 15

40 Enrichment of LMWPs using C18 absorbent / J. Wu et al. / Anal. Biochem. 398 (2010) 34–44

2-DE analysis of LMWPs

To verify C18 absorbent ability in enriching LMWPs, 2-DE anal-ysis was performed on three independent replicates of raw serum,unbound fractions, and eluted fractions. The distinctly differentprotein patterns indicated a significant removal of HMWPs andenrichment of LMWPs (Fig. 7). The detectable LMWP spot numbersbefore and after treatment were compared (Table 3). The meandetectable protein spot numbers were raised from 134 to 307 afterthe treatment, and the sensitivity of identified LMWPs also im-proved remarkably. For example, myeloid-related protein (MRP)family members, which were immune system-related proteins atthe 100 ng/ml level in healthy human serum [26], were hard to dis-play on a two-dimensional gel. In this experiment, two MRP pro-teins were first identified directly from the two-dimensional gel.The identification of MRP-8 is shown in Supplement Fig. 2 (seesupplementary material). Its confidential results also benefitedfrom the enrichment.

Quantification of LMWPs by ICAT labeling and LC–MS/MS

The ICAT labeling and LC–MS/MS method was used to quantifythe fold change of specific proteins. Sixteen proteins were confi-

Fig. 7. 2-DE protein profile of the raw serum and its unbound and eluted fractions. Seruserum fraction, and eluted serum fraction were dried in vacuum. Then 100 lg of each wPAGE).

dentially identified from detected peak pairs. Their peak areaswere individually calculated and compared. Their mean changewas 20.9-fold (Table 4). The peak pair of apolipoprotein C-III is alsoshown in Fig. 8.

Identification of LMWPs by LC–LTQ–Orbitrap MS/MS

Raw and treated serum were separated by Tricine–Tris SDS–PAGE. Protein bands (<30 kDa) were cut and LMWPs were identi-fied from raw and treated serum. A total of 47 LMWPs were iden-tified from raw serum (listed in Supplement Table 1 in thesupplementary material), and 150 LMWPs were identified fromtreated serum (listed in Supplement Table 2). More than 110LMWPs were identified from treated serum (listed in Table 5). It

m was treated by C18 absorbent as shown in the procedure. Raw serum, unboundas analyzed by 2-DE (pH 3–10 linear IEF strips, 65,000 V h, 15% Tricine–Tris SDS–

Table 4Fold change of serum LMWPs by ICAT labeling and LC MS/MS analysis.

Protein name Theoreticalmolecularweight (kDa)

Peptidesfor MS/MS

Ratio of average ± SD(after/before treatment)

Albumin fragment 71.3 6 8.11 ± 0.058Hemopexin 29.1 1 28.25Apolipoprotein B 28.3 1 19.56Apolipoprotein A-I 28.1 5 9.85 ± 0.018Apolipoprotein A-III 24.5 2 15.89 ± 0.098Transthyretin 16.0 4 12.83 ± 0.053Apolipoprotein D 14.1 1 14.00Serum amyloid A1 13.6 3 21.07 ± 0.061Serum amyloid A2 13.6 3 26.41 ± 0.14MRP-14 13.3 2 26.86 ± 0.014Hemoglobin 11.2 4 7.97 ± 0.027MRP-8 10.9 2 32.07 ± 0.035Apolipoprotein C-III 8.8 3 41.20 ± 0.064Apolipoprotein C-I 8.6 1 30.55Ubiquitin 8.4 1 18.57Apolipoprotein C-II 8.3 1 21.06

Enrichment of LMWPs using C18 absorbent / J. Wu et al. / Anal. Biochem. 398 (2010) 34–44 41

was noticed that 7 proteins were identified in raw serum, whereasthey failed to be identified in treated serum.

Discussion

Serum LMWPs are likely to contain abundant and interestinginformation that is closely related to the body physiological andpathological situations. Several methods have been introduced toenrich LMWPs, including the immunoaffinity and ultrafiltrationmethods. However, both of these methods could not effectively re-move HMWPs and enrich LMWPs that bind to HMWP carriers.

Here we have demonstrated a selective method to enrich low-abundance serum LMWPs. The enriching ability of the C18 absor-bent method was represented in three aspects. First, more than99% of HMWPs could be removed after the treatment (Table 2).Not only HSA and IgG but also other abundant HMWPs (>30 kDa)could be removed at the same time. The remaining HMWPs wereless than 5% in the eluted low-molecular-weight fractions anddid not disturb the detection of LMWPs. Second, the strong dena-tured conditions could break most protein–protein interactions

Fig. 8. Apolipoprotein C-III peak pair in ICAT labeling and LC–MS/MS quantification analyheavy chain (treated) was approximately 1:6, whereas the whole peak area ratio was 1

and reduce the loss of LMWPs that bound to HMWP carriers.LMWPs that bound to HSA or IgG could be separated and enrichedby the C18 absorbent method (Fig. 6). Third, for 1 ml of absorbent,the loading serum volume was 1 ml and the total protein contentin the eluted fraction was 0.9% (�400 lg), which could be totallyapplied to one 2-DE separation or LC–MS identification. In addi-tion, the C18 absorbent method also showed high efficiency. Be-cause the whole procedure was run under urea/DTTenvironment, which did not introduce salt or contaminants, no ex-tra desalting steps were needed before 2-DE analysis. The separa-tion and enrichment were very simple, and the process could berun in-tubes without instrumental limits so that it could be line-arly extended for massive production.

Based on these findings, the detectable spot numbers, concen-tration limit, and MS identification assurance of serum LMWPshave improved significantly. After the enrichment, the detectableLMWP spot numbers on 2-DE gels were increased by 229% (Table 3)and many identified LMWPs were low-abundance proteins, such asMRP-8, MRP-14, CTAP-III (connective tissue-activating peptide III),and ubiquitin, that had never been identified from serum sampleby 2-DE gels before. The ICAT labeling and LC–MS/MS quantifica-tion results were similar, but the average fold change was less(�20-fold) because the fold change was beyond the linear rangeof the ICAT quantification method.

After the enrichment, more than 110 LMWPs (<30 kDa) wereidentified from treated serum compared with raw serum. We no-ticed that 7 proteins (indicated in Supplement Table 1) were iden-tified in raw serum, whereas they failed to be identified in treatedserum. One possible reason was that these proteins might stronglybind and deposit with absorbent in such denatured environment,perhaps due to hydrophobic absorbent media properties and dif-ferent protein characteristics. When using hydrophilic C18 absor-bent (e.g., C18 solid phase extraction [SPE] absorbent), thisproblem could be partially resolved.

Compared with the affinity and ultrafiltration methods, the ma-jor advantage of the C18 absorbent method is its ability for enrich-ing carrier binding LMWPs. Because nondenatured conditionscould not break most protein–protein interactions and intensivelydenatured conditions would not be compatible for most proteinseparation methods, this approach might apply a selective methodfor carrier binding LMWP analysis.

sis. The figured apolipoprotein C-III peak intensity ratio of light chain (untreated) to:41.2.

Table 5More serum LMWPs (<30 kDa) identified by LC–LTQ–Orbitrap MS/MS after enrichment using C18 absorbent.

Protein ID Protein name Molecular weight (kDa) Peptides for MS/MS

IPI00220827 Thymosin beta-10 5.03 2IPI00182289 40S ribosomal protein S29 6.68 1IPI00787441 7 kDa protein 7.40 6IPI00815642 TMSB4X protein 7.77 1IPI00017448 40S ribosomal protein S21 9.11 2IPI00006705 Uteroglobin 9.99 1IPI00887241 40S ribosomal protein S28 10.27 1IPI00789259 V1–13 protein 10.34 1IPI00007047 Protein S100-A8 10.83 1IPI00220362 10 kDa heat shock protein, mitochondrial 10.93 1IPI00453473 Histone H4 11.37 5IPI00382436 Ig lambda chain V-III region SH 11.39 1IPI00008527 60S acidic ribosomal protein P1 11.51 1IPI00382440 Ig lambda chain V-IV region Hil 11.52 1IPI00550020 Parathymosin 11.53 1IPI00385555 Ig kappa chain V-I region BAN 11.84 1IPI00827535 Prothymosin alpha 12.20 2IPI00293276 Macrophage migration inhibitory factor 12.48 1IPI00552943 V1–11 protein 12.48 2IPI00830044 Uncharacterized protein ENSP00000374806 12.57 1IPI00014165 Histone H2A 12.65 1IPI00829947 13 kDa protein 13.05 1IPI00027462 Protein S100-A9 13.24 1IPI00552578 Serum amyloid A protein 13.53 5IPI00004656 Beta-2 microglobulin 13.71 3IPI00012750 40S ribosomal protein S25 13.74 1IPI00239077 Histidine triad nucleotide binding protein 1 13.80 1IPI00515061 Histone H2B type 1-J 13.90 7IPI00026272 Histone H2A type 1-B/E 14.14 4IPI00013917 40S ribosomal protein S12 14.53 3IPI00219219 Galectin-1 14.72 2IPI00219153 60S ribosomal protein L22 14.79 1IPI00419833 Histone H2B type 1-K 14.84 3IPI00010153 60S ribosomal protein L23 14.87 1IPI00908931 cDNA FLJ52456, moderately similar to programmed cell death protein 5 15.00 1IPI00216691 Profilin-1 15.05 2IPI00917298 60S ribosomal protein L31 15.12 1IPI00395998 60S ribosomal protein L32 15.86 1IPI00794659 cDNA FLJ58953, highly similar to 40S ribosomal protein S20 16.01 2IPI00473011 Hemoglobin subunit delta 16.06 9IPI00215780 40S ribosomal protein S19 16.06 1IPI00335168 Myosin light polypeptide 6 16.93 3IPI00798011 Uncharacterized protein SUMO3 16.96 1IPI00794543 cDNA FLJ75174, highly similar to Homo sapiens calmodulin 1 (phosphorylase kinase, delta), mRNA 17.16 3IPI00221089 40S ribosomal protein S13 17.22 1IPI00479997 Stathmin 17.30 2IPI00103142 NudC domain-containing protein 2 17.68 1IPI00013296 40S ribosomal protein S18 17.72 2IPI00024933 60S ribosomal protein L12 17.82 3IPI00412987 Glia maturation factor b 18.11 1IPI00433834 60S ribosomal protein L26 18.25 2IPI00025091 40S ribosomal protein S11 18.43 2IPI00012011 Cofilin-1 18.50 4IPI00473014 Destrin 18.51 1IPI00413344 Cofilin-2 18.74 1IPI00887504 60S ribosomal protein L21 18.88 1IPI00914529 cDNA FLJ57954, highly similar to 60S ribosomal protein L28 19.15 1IPI00011698 Histone deacetylase complex subunit SAP18 19.53 1IPI00873403 60S ribosomal protein L24 19.57 1IPI00375531 Nucleoside diphosphate kinase A 19.65 4IPI00895806 cDNA FLJ77718 19.69 1IPI00384857 Hematological and neurological expressed 1 protein 19.92 1IPI00376005 Eukaryotic translation initiation factor 5A-1 20.17 2IPI00376798 60S ribosomal protein L11 20.25 1IPI00556287 Putative uncharacterized protein 20.67 1IPI00026202 60S ribosomal protein L18a 20.76 1IPI00297579 Chromobox protein homolog 3 20.81 1IPI00006662 Apolipoprotein D 21.28 2IPI00644171 Putative uncharacterized protein RPL17P34 21.46 1IPI00215719 60S ribosomal protein L18 21.63 1IPI00433029 Insulin-like growth factor IB 21.84 4IPI00031691 60S ribosomal protein L9 21.86 1IPI00024915 Peroxiredoxin-5, mitochondrial 22.03 1IPI00478287 Putative uncharacterized protein ENSP00000352132 22.11 3IPI00221035 Transcription factor BTF3 22.17 1IPI00217466 Histone H1.3 22.35 4

42 Enrichment of LMWPs using C18 absorbent / J. Wu et al. / Anal. Biochem. 398 (2010) 34–44

Table 5 (continued)

Protein ID Protein name Molecular weight (kDa) Peptides for MS/MS

IPI00217468 Histone H1.5 22.58 1IPI00221088 40S ribosomal protein S9 22.59 1IPI00016608 Transmembrane emp24 domain-containing protein 2 22.76 1IPI00008433 40S ribosomal protein S5 22.88 3IPI00025329 60S ribosomal protein L19 23.47 1IPI00470528 60S ribosomal protein L15 24.15 1IPI00465361 60S ribosomal protein L13 24.26 2IPI00887678 Peptidyl-prolyl cis–trans isomerase A 24.38 2IPI00178440 Elongation factor 1-beta 24.76 1IPI00745660 IGL@ protein 24.86 5IPI00419258 High mobility group protein B1 24.89 3IPI00005981 Transgelin-3 24.89 1IPI00658130 IGL@ protein 25.02 9IPI00916434 Anti-(ED-B) scFV 25.15 3IPI00872430 40S ribosomal protein S8 25.19 1IPI00845354 IGKC protein 25.39 12IPI00005978 Splicing factor, arginine/serine-rich 2 25.48 2IPI00854806 IGKV1-5 protein 26.02 1IPI00795671 Putative uncharacterized protein RAN 26.22 1IPI00550731 Ig kappa chain V-II region RPMI 6410 26.23 2IPI00011253 40S ribosomal protein S3 26.69 3IPI00794402 Rho GDP dissociation inhibitor 1 27.52 2IPI00021263 14-3-3 Protein zeta/delta 27.75 5IPI00018146 14-3-3 Protein theta 27.76 2IPI00305380 Insulin-like growth factor-binding protein 4 27.93 1IPI00220642 14-3-3 Protein gamma 28.30 3IPI00010779 Tropomyosin alpha-4 chain 28.52 6IPI00021840 40S ribosomal protein S6 28.68 1IPI00021700 Proliferating cell nuclear antigen; cyclin 28.77 1IPI00549725 Phosphoglycerate mutase 1 28.80 3IPI00878611 cDNA FLJ57507, highly similar to RAN-specific GTPase-activating protein 28.84 1IPI00218319 Tropomyosin alpha-3 chain 29.03 8IPI00000816 14-3-3 Protein epsilon 29.17 1IPI00419880 40S ribosomal protein S3a 29.95 2

Enrichment of LMWPs using C18 absorbent / J. Wu et al. / Anal. Biochem. 398 (2010) 34–44 43

Although the separation mechanism of the C18 absorbent meth-od is not clear, we verified that the size of denatured proteins andthe pore size of C18 absorbent might be key to the separationmechanism. As an approach, we compared the selectivity of C18

absorbent with different pore sizes. It was shown that the molecu-lar weight selectivity of absorbent changed linearly with the poresize (Fig. 3). At the same time, we found that the RG values of pro-teins under urea/DTT denatured conditions were also linear withtheir molecular weights. This finding was different from that inKohn and coworkers’ report [27]. For no reducing agent was usedin their experiment, all SAS bonds remained and proteins wouldkeep coiled forms. In this study, proteins would likely be in linearform because all S–S bonds were broken. Meanwhile, the proteins’hydrophobic characteristics would change with the collapse oftheir hydrophobic domains under urea/DTT environment; thehydrophobic selectivity would be weakened. With our results, wecan conclude that molecular size replaced hydrophobicity as themajor factor for selectivity under urea/DTT denatured conditions.In addition, we tested the effect of absorbent ligand length. Whenreplacing C18 with C4 or C8, no significant difference was noticed(data not shown). So, the length of ligand may correlate with theprotein binding ability but not their separation selectivity. We alsotested other absorbent types, such as Silica Gel 60 GF254 and octyl-Sepharose CL-4B, which are widely used in protein separations[28]. We found that Silica Gel 60 GF254 without hydrophobic ligandshowed no protein binding ability, whereas octyl-Sepharose CL-4Bwith octyl as hydrophobic ligand showed protein binding abilitybut no separation ability (Fig. 5A).

In conclusion, we have developed an effective in-tube chro-matographic absorbent method to enrich LMWPs. After being incu-bated with 100 Å C18 absorbent under urea/DTT denatured

conditions followed by 60% ACN/H2O elution, a solution with ahigh concentration of LMWPs (<30 kDa) could be eluted out,whereas carrier binding LMWPs were enriched. Taking our findingstogether, we provide a useful method in enriching LMWPs either inserum or in cell and tissue samples.

Acknowledgments

We thank Rong Zheng (Department of Chemistry, Peking Uni-versity) for the collection and analysis of static laser light scatter-ing data. This work was supported by grants from the NationalKey Basic Research Program of China (2010CB912200 and2006CB910103) and the National Natural Science Foundation ofChina (90919023 and 30970652).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ab.2009.10.047.

References

[1] N.L. Anderson, N.G. Anderson, The human plasma proteome: history, character,and diagnostic prospects, Mol. Cell. Proteomics 1 (2002) 845–867.

[2] S.P. Gygi, G.L. Corthals, Y. Zhang, Y. Rochon, R. Aebersold, Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology, Proc.Natl. Acad. Sci. USA 97 (2000) 9390–9395.

[3] J.C. Byrne, M.R. Downes, N. O’Donoghue, J.M. Fitzpatrick, M.J. Dunn, R.W.Watson, Fasting status as a consideration for human serum collection andpreparation prior to depletion and analysis, Electrophoresis 29 (2008) 3055–3059.

44 Enrichment of LMWPs using C18 absorbent / J. Wu et al. / Anal. Biochem. 398 (2010) 34–44

[4] M. Ramstrom, A. Zuberovic, C. Gronwall, J. Hanrieder, J. Bergquist, S. Hober,Development of affinity columns for the removal of high-abundance proteinsin cerebrospinal fluid, Biotechnol. Appl. Biochem. 52 (2009) 159–166.

[5] B.A. Lollo, S. Harvey, J. Liao, A.C. Stevens, R. Wagenknecht, R. Sayen, J. Whaley,F.G. Sajjadi, Improved two-dimensional gel electrophoresis representation ofserum proteins by using ProtoClear, Electrophoresis 20 (1999) 854–859.

[6] N. Ahmed, G. Barker, K. Oliva, D. Garfin, K. Talmadge, H. Georgiou, M. Quinn, G.Rice, An approach to remove albumin for the proteomic analysis of lowabundance biomarkers in human serum, Proteomics 3 (2003) 1980–1987.

[7] H.M. Georgiou, G.E. Rice, M.S. Baker, Proteomic analysis of human plasma:failure of centrifugal ultrafiltration to remove albumin and other highmolecular weight proteins, Proteomics 1 (2001) 1503–1506.

[8] L.F. Steel, M.G. Trotter, P.B. Nakajima, T.S. Mattu, G. Gonye, T. Block, Efficientand specific removal of albumin from human serum samples, Mol. Cell.Proteomics 2 (2003) 262–270.

[9] K. Bjorhall, T. Miliotis, P. Davidsson, Comparison of different depletionstrategies for improved resolution in proteomic analysis of human serumsamples, Proteomics 5 (2005) 307–317.

[10] E.F. Petricoin, C. Belluco, R.P. Araujo, L.A. Liotta, The blood peptidome: a higherdimension of information content for cancer biomarker discovery, Nat. Rev.Cancer 6 (2006) 961–967.

[11] R.G. Harper, S.R. Workman, S. Schuetzner, A.T. Timperman, J.N. Sutton, Low-molecular-weight human serum proteome using ultrafiltration, isoelectricfocusing, and mass spectrometry, Electrophoresis 25 (2004) 1299–1306.

[12] R.S. Tirumalai, K.C. Chan, D.A. Prieto, H.J. Issaq, T.P. Conrads, T.D. Veenstra,Characterization of the low molecular weight human serum proteome, Mol.Cell. Proteomics 2 (2003) 1096–1103.

[13] W.W. Jung, S. Phark, S. Oh, J.Y. Khim, J. Lee, M.H. Nam, J.B. Seo, S.Y. Park, E. Jo, S.Choi, Z. Zheng, J.Y. Lee, M. Lee, E. Lee, D. Sul, Analysis of low molecular weightplasma proteins using ultrafiltration and large gel two-dimensionalelectrophoresis, Proteomics 9 (2009) 1827–1840.

[14] R. Kay, C. Barton, L. Ratcliffe, B. Matharoo-Ball, P. Brown, J. Roberts, P. Teale, C.Creaser, Enrichment of low molecular weight serum proteins usingacetonitrile precipitation for mass spectrometry based proteomic analysis,Rapid Commun. Mass Spectrom. 22 (2008) 3255–3260.

[15] J. He, Y. Liu, S. He, Q. Wang, H. Pu, J. Ji, Proteomic analysis of a membraneskeleton fraction from human liver, J. Proteome Res. 6 (2007) 3509–3518.

[16] M. Fountoulakis, J.F. Juranville, D. Roder, S. Evers, P. Berndt, H. Langen,Reference map of the low molecular mass proteins of Haemophilus influenzae,Electrophoresis 19 (1998) 1819–1827.

[17] X. Peng, Y. Wu, J. Chen, S. Wang, Proteomic approach to identify acute phaseresponse-related proteins with low molecular weight in loach skin followinginjury, Proteomics 4 (2004) 3989–3997.

[18] D.F. Meyer, A.S. Nealis, K.R. Bruckdorfer, S.J. Perkins, Characterization of thestructure of polydisperse human low-density lipoprotein by neutronscattering, Biochem. J. 310 (1995) 407–415.

[19] Z. Rui, J. Jian-Guo, T. Yuan-Peng, P. Hai, R. Bing-Gen, Use of serologicalproteomic methods to find biomarkers associated with breast cancer,Proteomics 3 (2003) 433–439.

[20] A. Lu, J.R. Wisniewski, M. Mann, Comparative proteomic profiling ofmembrane proteins in rat cerebellum, spinal cord, and sciatic nerve, J.Proteome Res. 8 (2009) 2418–2425.

[21] Y. Liu, J. He, S. Ji, Q. Wang, H. Pu, T. Jiang, L. Meng, X. Yang, J. Ji, Comparativestudies of early liver dysfunction in senescence-accelerated mouse usingmitochondrial proteomics approaches, Mol. Cell. Proteomics 7 (2008) 1737–1747.

[22] W. Yang, P. Liu, Y. Liu, Q. Wang, Y. Tong, J. Ji, Proteomic analysis of ratpheochromocytoma PC12 cells, Proteomics 6 (2006) 2982–2990.

[23] J.X. Yan, R. Wait, T. Berkelman, R.A. Harry, J.A. Westbrook, C.H. Wheeler, M.J.Dunn, A modified silver staining protocol for visualization of proteinscompatible with matrix-assisted laser desorption/ionization and electrosprayionization–mass spectrometry, Electrophoresis 21 (2000) 3666–3672.

[24] F. Gharahdaghi, C.R. Weinberg, D.A. Meagher, B.S. Imai, S.M. Mische, Massspectrometric identification of proteins from silver-stained polyacrylamidegel: a method for the removal of silver ions to enhance sensitivity,Electrophoresis 20 (1999) 601–605.

[25] N. Guerreiro, J.W. Redmond, B.G. Rolfe, M.A. Djordjevic, New Rhizobiumleguminosarum flavonoid-induced proteins revealed by proteome analysis ofdifferentially displayed proteins, Mol. Plant Microbe Interact. 10 (1997) 506–516.

[26] T. Bogumil, P. Rieckmann, B. Kubuschok, K. Felgenhauer, W. Bruck, Serumlevels of macrophage-derived protein MRP-8/14 are elevated in activemultiple sclerosis, Neurosci. Lett. 247 (1998) 195–197.

[27] J.E. Kohn, I.S. Millett, J. Jacob, B. Zagrovic, T.M. Dillon, N. Cingel, R.S. Dothager,S. Seifert, P. Thiyagarajan, T.R. Sosnick, M.Z. Hasan, V.S. Pande, I. Ruczinski, S.Doniach, K.W. Plaxco, Random-coil behavior and the dimensions of chemicallyunfolded proteins, Proc. Natl. Acad. Sci. USA 101 (2004) 12491–12496.

[28] H. Bunger, L. Kaufner, U. Pison, Quantitative analysis of hydrophobicpulmonary surfactant proteins by high-performance liquid chromatographywith light-scattering detection, J. Chromatogr. A 870 (2000) 363–369.