Elsevier

Talanta

Volume 182, 15 May 2018, Pages 333-339
Talanta

Modulating the protein content of complex proteomes using acetonitrile

https://doi.org/10.1016/j.talanta.2018.01.057Get rights and content

Highlights

  • Acetonitrile is presented as a modulator of protein content in complex proteomes.

  • It can be used as a tool to facilitate profiling of patients.

  • Time, cost, sample requirement, reagent consumption, energy requirements and waste production is minimized.

Abstract

In this work we present acetonitrile as a tool to modulate the dynamic range of the proteome of complex samples. Different concentrations of acetonitrile ranging from 15% v/v to 65% v/v were used to modulate the protein content of serum samples from healthy people and patients with lymphoma and myeloma. We show that the proteome above 70 kDa is pelleted as a function of the concentration of acetonitrile and that profiling with PCA or Clustering is only possible using the supernatants obtained for concentrations of acetonitrile higher than 45% v/v or the pellets for concentrations of acetonitrile of 35% and 45%. The differentiation and classification of the three groups of sera samples (healthy, lymphoma and myeloma) were possible using acetonitrile at 55% v/v concentration. This work opens new avenues for the application of acetonitrile as a cost-effective tool in proteomics applications.

Introduction

Plasma and serum are complex proteomes interrogated by researchers and physicians for diagnostics and prognostic purposes because both are composed of thousands of proteins that contain vital information about the health status of individuals. Variations in proteins and their concentrations can be linked to diseases or to the organism's response to drugs [1], [2], [3], [4]. One of the challenges of studying complex proteomes is the range in proteins concentration, which for serum and plasma is higher than ten orders of magnitude [1], [2], [3], [4]. Some proteins, called high-abundance proteins, HAP, are present at levels of gram per litter and they constitute the vast majority of the total protein mass. In fact as little as 12 proteins constitute around 95% of the total mass of proteins in plasma and serum [5]. On the contrary, some proteins are present at very low levels when compared with the HAP, and they can be present temporarily as a result of a disease or a therapy. These low-abundance proteins, LAP, carrying great diagnostic potential, are often obscured by the presence of high-abundance serum proteins because the dynamic range of concentrations surpasses the capabilities of existing separation and analysis techniques. Therefore, a large number of standard approaches for reducing the complexity of plasma and serum proteomes have been proposed and they can be assigned into three approaches; immunodepletion, affinity enrichment, and fractionation [1]. There are different commercial approaches to compress the dynamic range of the proteins by depleting the high-abundance ones. Thus, The ProteoPrep 20 Plasma immunodepletion kit, from Sigma-Aldrich [6], has been developed for the removal of the 20 high-abundance proteins and the Proteominer, from Bio-Rad, is based on treating complex protein samples with a library of hexapeptides bound to chromatographic supports [7]. Protein equalization for biomarker discovery has been performed with success in sera samples and peritoneal liquid using dithiothreitol by our team [8], [9]. Furthermore, protein depletion with acetonitrile was also proven to be very useful [10]. In the latter case, however, an exhaustive study on the effects of the ACN concentration in the process of depletion has not been done yet, to the best of our knowledge. In this work we demonstrated that acetonitrile works as a modulator of the protein content of sera samples and that this property can be used for reducing the complexity of the serum proteome. As proof of concept, matrix-assisted laser desorption ionization mass spectrometry-based profiling of ACN-depleted sera samples of healthy volunteers and myeloma and lymphoma patients is presented.

Section snippets

Reagents

All reagents used were HPLC grade or electrophoresis grade. Albumin, from bovine serum (BSA), (N, N, N, N′-tetramethylethylene-diamine (TMED), glycine, β-mercaptoethanol, glycerol 86–88%, Bradford reagent, coomassie blue G-250, tris–base, DL-dithiothreitol (DTT), iodoacetamide (IAA), acrylamide/bis- acrylamide 30% solution (37.5:1) were purchased from Sigma-Aldrich (Basel, Switzerland). Formic acid, ammonium bicarbonate (Ambic), ammonium persulfate and α-cyano-4-hydroxycinnamic acid were

Results and discussion

The effect of the variation in the concentration (%v/v) of acetonitrile over the serum proteins levels may be seen in Fig. 1A, showing the gel electrophoresis (1-D) profiles of the serum supernatant and serum pellet after protein depletion. Thus, it may be seen that, as the ACN concentration is increased, so the protein concentration in the supernatant decreases steadily, but continuously, until the concentration of 45% is reached. Then, when the ACN concentration is increased up to 55% (v/v),

Conclusions

ACN can be used as a successful modulator of the protein content of samples with complex proteomes, such as serum, because it works as an efficient compressor of the concentration dynamic range of high-abundance proteins. The latter is very important for successful profiling of samples using MALDI-based MS, as demonstrated in this work with healthy, myeloma and lymphoma samples. This work opens new avenues for the application of acetonitrile as a cost-effective tool in proteomics applications,

Acknowledgements

The PROTEOMASS Scientific Society is acknowledged by the funding provided to the Laboratory for Biological Mass Spectrometry - Isabel Moura and to this work. Authors acknowledge the funding provided by UCIBIO, Unidade de Ciências Biomoleculares Aplicadas, which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728) and to the Associate Laboratory for Green Chemistry LAQV which is financed

References (13)

There are more references available in the full text version of this article.

Cited by (3)

  • Europium coordination polymer particles based electrospun nanofibrous film for point-of-care testing of copper (II) ions

    2021, Talanta
    Citation Excerpt :

    The sample analysis experimental protocol was approved by the institutional committee of Southwest University (approval number yxy202107). Three groups of normal people's blood were centrifuged to obtain the upper serum, and then the protein in the serum was removed by the acetonitrile precipitation method [33]. Take 100 μL of the serum and 100 μL of Cu2+ solution with different concentrations (100 μM, 250 μM, 400 μM) into a 1.5 mL EP tube which containing 400 μL of BR buffer (pH 3.5).

  • Ultrasonic-assisted extraction and digestion of proteins from solid biopsies followed by peptide sequential extraction hyphenated to MALDI-based profiling holds the promise of distinguishing renal oncocytoma from chromophobe renal cell carcinoma

    2020, Talanta
    Citation Excerpt :

    Because the samples used in this case were not plasma, but extracts obtained from tissue biopsies, we first performed a set of experiments to assess the effects of ACN on protein depletion. The ACN concentrations selected were 20% (v/v) and 45% (v/v) based on our experience with this sample treatment [13,21]. As can be seen in Fig. 2A, protein depletion with 45% (v/v) ACN concentration renders a pellet with higher concentration of proteins reflecting their depletion from the supernatant.

View full text