Henning 2015

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Received: 05-Jun-2014; Revised: 15-Dec-2014; Accepted: 28-Jan-2015

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Technical brief

An alternative method for serum protein depletion/enrichment by precipitation at

mildly acidic pH values and low ionic strength

Ann-Kristin Henning1, Dirk Albrecht

2 , Katharina Riedel

2, Thomas C. Mettenleiter

1, Axel

Karger1*

1 Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut,

Südufer 10, 17493 Greifswald-Insel Riems, Germany

2 Institute for Microbiology, Ernst-Moritz-Arndt-University Greifswald, Friedrich-Ludwig-

Jahn-Straße 15, 17487 Greifswald, Germany

* Corresponding author.

Dr. Axel Karger

Postal address: Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald – Insel Riems,

Germany

Telephone number: +49 3835171247.

E-mail address: [email protected]

Keywords: equalization, ion strength, precipitate, proteome, serum

Total number of words: 2959

mailto:[email protected]



Abstract

Serum proteome analysis is severely hampered by the extreme dynamic range of protein

concentrations, but tools for the specific depletion of highly abundant serum proteins lack for

most farm and companion animals. A well-established alternative strategy to reduce the

dynamic range of plasma protein concentrations, treatment with combinatorial peptide ligand

libraries (CPLL), is generally applicable but requires large amounts of sample. Therefore,

additional depletion/enrichment protocols for plasma and serum samples from animals are

desirable. In this respect we have tested a protein precipitate that formed after withdrawal of

salt from human, bovine, or porcine serum at pH 4.2. The bovine sample was composed of

over 300 proteins making it a potential source for biomarker discovery. Precipitation was

highly reproducible and the concentrations of albumin and other highly abundant serum

proteins were strongly reduced. In comparison to CPLL-treatment, precipitation did not

introduce any selection bias based on hydrophathy or pI. However, the composition of both

preparations was partially complementary. Salt withdrawal at pH 4.2 is suggested as

additional depletion/enrichment strategy for serum samples. Also, we point out that the

removal of precipitates from serum samples under the described conditions bears the risk of

losing a valuable protein fraction.

Plasma and serum analysis is severely hampered by the extreme dynamic range of protein

concentrations which spans 10 or more orders of magnitude [1, 2]. According to Tirumalai et

al. [3] the 10 most abundant proteins in human plasma constitute approximately 90% of the

total protein content. The most abundant serum protein is albumin, accounting for more than

50% of its protein content [3].



To facilitate serum proteome analysis or biomarker studies two strategies have been

developed to reduce the range of protein concentrations. Either highly abundant proteins are

depleted by specific extraction or protein concentrations are equalized e.g. by solid phase

extraction using immobilized combinatorial peptide ligand libraries (CPLL). The first

approach relies on affinity extraction of the targeted proteins. Removal of albumin from

human sera by Cibacron dyes [4] has been a cornerstone of plasma protein analysis.

Chromatographic maxtrices for the immunodepletion of the most abundant proteins have

been developed for human sera but also for some small laboratory animals [5, 6]. All

depletion techniques bear the risk of unintentional loss of untargeted proteins that bind to the

matrix in an unspecific way or are ligands of the highly abundant targeted proteins [7]. The

second strategy, equalization of protein concentrations, can be implemented by extraction

with libraries of immobilized combinatorial hexapeptides [8] or by sample displacement

chromatography [9]. Both approaches use modified solid supports that are treated with excess

plasma proteins. Under conditions of saturation, low abundance proteins are enriched and

high abundance components with low affinity to the support can be removed by washing. As

for the most farm and companion animals tools for the specific depletion of abundant plasma

proteins, e.g. by immunodepletion, are not available, equalization remains as the only option

to prepare sera or plasmas for proteome analysis. A major drawback of equalization is the

need for large amounts of serum to achieve saturating conditions and acceptable protein

yields which, in our laboratory, are approximately 0.6% for bovine sera. Also, elution

conditions have been discussed in the literature as the gold standard for efficient elution,

boiling in 4% SDS and 25 mM DTT [10], requires the removal of SDS for some downstream

applications as e.g. 2DE. Therefore, broadly applicable alternative protocols for the reduction

of the protein concentration range in animal sera are highly desirable in the veterinary

medicine.



Studying the bovine serum proteome we had observed that salt removal under certain

experimental conditions lead to the precipitation of a protein fraction (Fig. 1) exhibiting

interesting features. The precipitation yields, 4% of the input protein, were very stable

irrespective of how the salt was withdrawn experimentally, by dilution or dialysis. The

protein pattern after SDS-PAGE was very reproducible and strongly reminiscent of CPLL-

extracted material (Fig. 2A) with well-balanced protein concentrations over a wide range of

molecular weights (Fig.2, Supporting Information Fig. 1 ). The protein pattern of the

remaining supernatant was very similar to that of raw serum, suggesting that the precipitate is

enriched in less abundant serum proteins (Fig. 2A). Finally, under the same experimental

conditions precipitations with similar appearance also formed from human and porcine sera

(Supporting Information, Fig. 2).

To characterize this precipitate, we optimized precipitation conditions, identified the

constituting proteins by mass spectrometry, analyzed their physico-chemical properties and

determined the depletion of the most abundant serum components using SDS-PAGE, 2DE

and quantitative nLC-MALDI-TOF/TOF MS. For comparison, a protein fraction obtained by

extraction with a commercially available CPLL was analyzed in parallel.

Serum samples were collected from healthy cows by standard procedures as described

elsewhere [11]. Equalization was performed using ProteoMiner beads (BioRad) as described

in the Supporting Information Material and Methods file. This preparation is referred to as

„equalized proteins‟.

Conditions for efficient precipitation were optimized with respect to pH and salinity (Fig. 1).

Variation of the pH at 1mM buffer concentration identified a pH of 4.2 to be optimal with

respect to precipitation yields and balanced protein concentrations (Figure 1A). At the

optimal pH, increasing buffer concentrations (Figure 1B) or addition of NaCl (Figure 1C)

gradually inhibited precipitation probably by a salting-in effect. For the following



experiments, the precipitate was prepared by dialysis (SnakeSkin Dialysis Tubing, 7000 Da

molecular weight cutoff; Thermo Scientific) against 1 mM Na2HPO4/citric acid (NPC) pH

4.2 for 24 h at 4 °C and recovered by centrifugation at 16,000 g for 10 min. It is referred to

simply as „precipitate‟. Very similar results were obtained when the precipitation experiments

were repeated with human or porcine sera (Supporting Information, Fig. 2). Replacement of

NPC by other buffer systems that are useful at pH 4.2 produced virtually the same

precipitates (not shown).

Comparison of the protein profiles of equalized and precipitated proteins after SDS-PAGE

revealed marked differences in protein composition (Fig. 2A). For a more detailed view 150

µg samples of raw serum, equalized proteins and precipitate were analyzed by 2DE. Pairwise

two-channel color overlay images of the 2D electrophoretic gels of raw, equalized and

precipitated proteins are shown in the Supporting Information, Fig. 3. A number of protein

spots were shared between both fractions but there also was remarkable complementarity.

Comparison with the 2DE of raw serum showed that albumin was efficiently depleted in both

preparations (Fig. 2B).

Reproducibility of precipitation was tested using a quantitative approach. Two independent

preparations of the precipitate from the same serum were trypsin digested, peptides labelled

with isotope-tagged dimethyl groups and 1:1 mixes analyzed by quantitative nLC-MALDI-

TOF/TOF MS as described in the Supporting Information Material and Method file. The

experiment was carried out in triplicate using sera from three different animals. The same

experimental scheme was applied to three 1:1 mixtures each of differentially labelled digests

of raw serum and of equalized proteins. The resulting relative protein abundances are

presented as quantile plots in Fig. 3A. In comparison with raw serum both treatments

introduced minimal additional experimental variation. However, relative abundances of

>95% of the identified proteins were within a 1.5 fold range after equalization and

precipitation, indicating reproducibility of both treatments was very similar and satisfactory.

The proteins identified in these nLC-MALDI-TOF/TOF MS experiments were used to

confirm the differences in protein composition between the equalized material and the

precipitate that were observed in the electrophoretic analysis (Fig. 2A, Supporting

Information Fig. 3). Panels of proteins identified in the three different preparations (raw

serum, equalized or precipitated proteins) are listed in the Supporting Information Table 1

and were compared in the Venn diagram [12] in Figure 3B. We observed some overlap but



also a substantial degree of complementarity that confirmed SDS-PAGE and 2DE and

indicated that equalization and precipitation may be used as complementary techniques for

serum analysis. Under the experimental conditions applied, a number of proteins were

reliably identified in the precipitate (three out of three replicates) that were not identified at

all in raw serum, e.g. coagulation factors V and X, complement factors H and I, protease

inhibitors like serpin A10, serpin F1, and inter-alpha-trypsin inhibitor heavy chain H3, and a

number of complement-related proteins like subcomponents r and s of complement

component 1, or the complement component 8 alpha and gamma chains.

The physicochemical properties of the proteins identified by nLC-MALDI-TOF/TOF MS in

the three replicates of the equalized and precipitated samples were analyzed. Particularly, the

grand averages of hydropathy (GRAVY) [13], molecular weights, and the isoelectric points

were calculated and compared to those of the bovine proteins currently annotated to the

“extracellular space” (GO-term GO:0005615) in the EMBL database [14], release 67

(Supporting Information Table 2), for details see the Supporting Information Material and

Methods file. Figure 3C shows the representation of experimentally identified proteins in the

two preparations over GRAVY, Mr and pI in comparison to the reference proteins. Over the

entire range of GRAVY values, distribution of experimentally identified proteins from both

preparations matched the expected values of the reference proteins indicating that neither

protocol introduced a hydrophobicity based selection bias. For both protocols, proteins with

Mr < 12,000 Da were found to be underrepresented, presumably as a consequence of the

dialysis step during sample preparation. Thus, both protocols may have to be adapted for

studies targeting low molecular weight serum proteins. This effect was slightly more

pronounced for the precipitated proteins. Profiles of both fractions over the pI were very

similar and indicated that proteins with pI > 8.0 were not adequately represented in neither

fraction. Most importantly, the profiles of both fractions were very similar showing that, in

comparison to equalization, precipitation did not introduce any notable bias preferring

proteins on basis of their physicochemical parameters, with the exception of a more

pronounced underrepresentation of smaller proteins.

Next, the depletion of individual highly abundant serum proteins by both treatments was

assessed. To this purpose tryptic digests of raw serum, equalized and precipitated proteins

were isotope labelled by reductive dimethylation [15] using conventional or deuterated

formaldehyde to introduce a 4 Da mass tag per dimethyl group. Peptides were mixed at 1:1

ratios and analyzed as described in the Supporting Information, Material and Methods. All

experiments were done in duplicate with reciprocal labeling using independent preparations

of one serum . For experimental details see the Material and Method file in the Supporting

Information.



Depletion or enrichment of proteins by precipitation was assessed in mixtures of isotope

labelled precipitates and raw serum preparations. Efficient depletion was found for some of

the most abundant serum proteins like albumin, immunoglobulins, apolipoprotein A-I,

serotransferrin, -2-HS-glycoprotein, or hemopexin, while a number of less abundant

proteins were enriched like e.g. some complement-related proteins. For the most efficiently

depleted or enriched proteins, detailed results are given in Supporting Information Table 4.

For direct comparison of the depletion efficiency of the most abundant proteins by

equalization and precipitation, 1:1 mixtures of both preparations were analyzed by

quantitative MS. In the following, abundance ratios of two experiments are given with ratios

below 1 indicating a more effective depletion by equalization than by precipitation and vice

versa. Only values within the measurable range of approximately 20-fold are specified.

Albumin was depleted with very similar efficiency (1.0/0.71) whereas immunoglobulins

(0.43/0.31), Alpha-2-macroglobulin (0.28/0.36), Complement C3 (0.39/0.42), and Inter-

alpha-trypsin inhibitor heavy chain H4 (0.30/0.65) were removed more efficiently by

equalization. However, Apolipoprotein A-I (>20/>20), and also the less abundant

apolipoproteins A-II (>20/>20), A-IV (>20/14), and C-III (9.4/9.5) were highly enriched by

equalization. Depletion of Serotransferrin (2.8/2.7) by precipitation was more efficient than

by equalization.

From these quantitative experiments we conclude that precipitation efficiently depletes some

of the most abundant serum proteins while other less abundant proteins are enriched.

Precipitation was beneficial for MS analysis as substantially more proteins could be identified

from precipitations than from raw serum preparations. The enrichment/depletion

characteristics of precipitation and equalization markedly differed so that both techniques

may be favorably combined.



Finally, in-depth proteome analysis of the precipitate was carried out using a nLC-MALDI-

TOF/TOF MS platform and a nLC-LTQ OrbitrapXL system. Before MS analysis, tryptic

peptides were fractionated by gel-free IEF to improve yields of identified proteins [11]. MS

data from 24 nLC runs (12 IEF fractions analyzed on each platform) were combined and a

joint list of identified proteins was compiled. For experimental details see the Supporting

Information Material and Methods file. In total, 306 proteins were identified on basis of a

minimum of 2 identified peptides per protein (Supporting Information Table 3). Gene

ontology (GO) analysis of the identified proteins using QickGO [16]

(http://www.ebi.ac.uk/QuickGO/GAnnotation) and Blast2GO software [17] showed that 184

(60.1%) identified proteins were annotated with GO-term GO:0005615 which is

recommended for plasma proteins. This is higher than we had observed in a recent study of

the bovine plasma proteome (41.7%) using equalized samples [11], indicating that numerous

typical serum proteins are efficiently precipitated under the described conditions.

In conclusion, we show that precipitation of serum proteins by salt withdrawal yields a

protein fraction which shows similarity to CPLL treated proteins. A number of highly

abundant serum proteins are efficiently depleted and thus protein identification by MS is

enhanced. Both treatments efficiently remove Albumin and Transferrin. Depletion and

enrichment of individual other highly abundant proteins is partially complementary. Protein

yields after precipitation are higher than after equalization making precipitation applicable in

cases where the serum samples are limited, e.g. when analyzing serum from smaller animals.

In-depth protein analysis showed that the precipitate is rich in proteins and the composition is

dominated by typical serum proteins. Precipitation is economic, rapid, and high-throughput

capable. No reagents, particularly detergents, are introduced that may be incompatible with

following analysis. We suggest precipitation as a species-unspecific protocol to reduce the

dynamic range of protein concentrations, not to replace but rather to complement CPLL

treatment in serum proteomic workflows. Also, we would like to point out that removal of

precipitations that occur under the specified conditions, e.g. when preparing serum samples

for ion exchange chromatography, may lead to the loss of this potentially valuable protein

fraction.

Acknowledgements

This study was supported by the Federal Ministry of Education and Research, Phenomics

Network of Excellence, Germany.

Conflict of interest statement

None of the authors of this paper has a financial or personal relationship with other people or

organizations that could inappropriately influence or bias the content of the paper.



References

[1] Anderson, N. L., Anderson, N. G., The human plasma proteome: history, character, and diagnostic prospects. Molecular & cellular proteomics : MCP 2002, 1, 845-867. [2] Mitchell, P., Proteomics retrenches. Nature biotechnology 2010, 28, 665-670. [3] Tirumalai, R. S., Chan, K. C., Prieto, D. A., Issaq, H. J., et al., Characterization of the low molecular weight human serum proteome. Molecular & cellular proteomics : MCP 2003, 2, 1096-1103. [4] Travis, J., Pannell, R., Selective removal of albumin from plasma by affinity chromatography. Clinica chimica acta; international journal of clinical chemistry 1973, 49, 49-52. [5] Yadav, A. K., Bhardwaj, G., Basak, T., Kumar, D., et al., A systematic analysis of eluted fraction of plasma post immunoaffinity depletion: implications in biomarker discovery. PLoS One 2011, 6, e24442. [6] Linke, T., Doraiswamy, S., Harrison, E. H., Rat plasma proteomics: effects of abundant protein depletion on proteomic analysis. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 2007, 849, 273-281. [7] Bellei, E., Bergamini, S., Monari, E., Fantoni, L. I., et al., High-abundance proteins depletion for serum proteomic analysis: concomitant removal of non-targeted proteins. Amino acids 2011, 40, 145-156. [8] Thulasiraman, V., Lin, S., Gheorghiu, L., Lathrop, J., et al., Reduction of the concentration difference of proteins in biological liquids using a library of combinatorial ligands. Electrophoresis 2005, 26, 3561-3571. [9] Josic, D., Breen, L., Clifton, J., Gajdosik, M. S., et al., Separation of proteins from human plasma by sample displacement chromatography in hydrophobic interaction mode. Electrophoresis 2012, 33, 1842-1849. [10] Candiano, G., Dimuccio, V., Bruschi, M., Santucci, L., et al., Combinatorial peptide ligand libraries for urine proteome analysis: investigation of different elution systems. Electrophoresis 2009, 30, 2405-2411. [11] Henning, A. K., Groschup, M. H., Mettenleiter, T. C., Karger, A., Analysis of the bovine plasma proteome by matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry. Vet J 2014, 199, 175-180. [12] Micallef, L., Rodgers, P., eulerAPE: drawing area-proportional 3-Venn diagrams using ellipses. PLoS One 2014, 9, e101717. [13] Kyte, J., Doolittle, R. F., A simple method for displaying the hydropathic character of a protein. J Mol Biol 1982, 157, 105-132. [14] Flicek, P., Ahmed, I., Amode, M. R., Barrell, D., et al., Ensembl 2013. Nucleic Acids Res 2013, 41, D48-55. [15] Boersema, P. J., Raijmakers, R., Lemeer, S., Mohammed, S., Heck, A. J., Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nature protocols 2009, 4, 484-494. [16] Binns, D., Dimmer, E., Huntley, R., Barrell, D., et al., QuickGO: a web-based tool for Gene Ontology searching. Bioinformatics 2009, 25, 3045-3046. [17] Conesa, A., Gotz, S., Garcia-Gomez, J. M., Terol, J., et al., Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005, 21, 3674-3676. [18] Hortin, G. L., Sviridov, D., Anderson, N. L., High-abundance polypeptides of the human plasma proteome comprising the top 4 logs of polypeptide abundance. Clin Chem 2008, 54, 1608-1616.



Fig. 1 Precipitation of serum proteins under variation of pH (A), buffer concentration (B) and

salinity (C). (A) Precipitates formed after dialysis in a wide pH range, but the most balanced

band intensities and the highest protein yields were observed after dialysis against 1 mM

NPC buffer at pH 4.2. Precipitation yields at pH 4.2 decreased with increasing buffer

concentrations (B) or addition of NaCl to the 1 mM NPC buffer, pH 4.2, indicating a salting-

in effect. M: molecular weight marker.



Fig.2 Comparison of the protein patterns of raw (“R”), equalized (“CPLL”) and precipitated

(“PR”) serum after SDS-PAGE (A) and 2DE (B). In panel A, 10 µg of the indicated fractions

or the supernatant of the precipitate (“Sn”) were analyzed. Note the similarity of raw serum

and supernatant of the precipitation indicating that the precipitate may be rich in low-

abundance proteins. The asterisks mark serum albumin. Panel B shows the corresponding 2D

electrophoretic patterns of 150 µg aliquots. M: molecular weight marker.



Fig. 3 Comparison of equalized and precipitated proteins by nLC-MALDI TOF/TOF MS

analysis. Isotope-labelled peptide preparations of raw serum, equalized or precipitated

samples were mixed in 1:1 ratios and quantitated by MS. The distributions of the relative

abundances of identified proteins were calculated and are presented as quantile plots (A) over

the medians of the isotope ratios of the respective protein-specific peptides. Horizontal dotted

lines represent the 1, 5, 10, 90, 95, and 99 % quantiles respectively. Distributions of relative

abundances are centered around 1.0 and very narrow indicating a low degree of experimental

variation of raw, equalized (dotted) and precipitated (dashed) protein preparations. (B) Area-

proportional Venn diagram representing the number of proteins reliably (three out of three

replicates) identified in raw, equalized, and precipitated serum. (C) To test for any bias

introduced by equalization (plain lines) or precipitation (dashed lines) with respect to

hydrophobicity (GRAVY), molecular weight (log10(MW)), or charge (pI), these parameters

of the identified proteins were compared with those calculated for bovine serum proteins

listed in the EMBL database. For details see the Supporting Information Material and

Methods file. Positive and negative values indicate over- and underrepresentation of the

identified proteins in the respective parameter range in comparison to the reference proteins.

Binary logarithms of the ratios are given.

Henning 2015

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