One of the dilemmas of protein characterization in biological samples is the dominance of high abundance proteins which complicate the detection and analysis of low abundance proteins (LAPs). In the case of blood plasma, a total of 22 proteins, albumin, total IgG, transferrin, fibrinogen, IgA, alpha-2-macroglobulin, IgM, alpha-1-anti-trypsin, C3 complement, haptoglobulin, alpha-1-acid glycoprotein, apolipoprotein-B, apolipoprotein-A1, lipoprotein a, factor H, ceruloplasmin, C4 complement, complement factor B, pre-albumin, C9 complement, C1q complement, and C8 complement, comprise 99% of the total human plasma protein content, while hundreds of other proteins contribute to the remaining 1% [3, 4]. Recent studies have demonstrated that characterization of LAPs can be performed by biochemical and molecular biology methods, such as mass spectrometry (MS), Western blotting (WB), nuclear magnetic resonance (NMR), or single-molecule peptide sequencing [5, 6], either with the presence of high abundance proteins or with the enrichment of LAPs.

Several methods for LAP enrichment have recently been developed (Table 1). A novel approach based on metabolic glycoprotein labelling and click chemistry for the enrichment of glycoproteins, named "improved Secretome Protein Enrichment with Click Sugars" - iSPECS, has been presented during the SFN 2018 meeting [7]. Blood proteins can also be enriched in vivo via the injection and collection of liposomes [8].

A new isolation method based on collagenase treatment has been developed in order to improve MS analysis of LAPs [9]. The number of detected LAPs was increased by 1.3-2.2 fold, depending on the tissue. This technique has been successfully used for characterization of LAPs in adipose tissue, bone and tendon. In addition, the selected reaction monitoring assay has been used to improve the detection of the LAPs of bacterial origin by MS [23].

Also, another approach for the characterization of LAPs based on the proteomic reactor combined with pH fractionation has previously been described [17]. The proteomic reactor has been generated using a strong anion exchange. The obtained pH fractions have shown enhanced protein characterization with approximately 50% of fractionated proteins being LAPs as detected by mass spectrometry.

Enhanced resolution of LAPs can also be achieved by isopropanol extraction [13]. This method has been used to detect LAPs in soybean seeds. The isopropanol-based extraction has been followed by polyacrylamide gel electrophoresis (PAGE), which indicated that isopropanol at concentrations higher than 30% would provide significant enrichment of LAPs. Protein identification has been performed by mass spectrophotometry (MALDI-TOF-MS).

Furthermore, another fast and effective approach, which combines polyethylene glycol separation and immunoaffinity-based depletion, has recently been published [15]. Multiple LAPs with concentrations lower than 100 ng/mL have been identified in human serum using this technique, suggesting significant enhancement of LAP characterization. This method has raised the detection of LAPs by 43%.

Depletion of highly abundant proteins is a logical and effective approach to promote characterization of LAPs. Kim et al have applied protamine sulfate precipitation to remove abundant proteins from total seed proteins [16]. The analysis with SDS-PAGE or Western blotting of 500 μg of protein samples has shown enhanced spots in protamine sulfate-treated fraction compared to the untreated total fraction. The 0.05% of protamine sulfate was enough to remove major abundant proteins.

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Figure 1. Signal amplification using gold nanoparticles. The amplification strategy consisted of: (a) generation of aptamer-bound gold substrate, (b) generation of mass tagged mAuNP, and (c) characterization of proteins by mass spectrometry [1].

An additional platform for characterization of LAPs in biological fluids using magnetic nanoparticles (MNP) has recently been developed (Fig. 1) [2]. The method based on EDTA-functionalized nanoparticles has been applied to protein analysis of significant urine volumes and showed higher protein enrichment and a moderate cost in comparison to ultrafiltration techniques.

New methods have recently been developed to remove high abundant proteins (HAP) and enrich LAPs in biological liquids. Both MNPs and treatment with dithiothreitol and acetonitrile allowed efficient depletion of several HAPs, such as albumin and others, and amplification of the LAPs [24].

In addition, monodisperse MNPs, which were generated by gold and surfactant covering to enhance hydrophilic characteristics, have been suggested to be a very effective technique for the enrichment of LAPs [14]. The authors have tested this approach for the analysis of C-reactive protein. Notably, the sensitivity limit of monodisperse MNPs was about 1 ng per mL of the testing volume, while standard clinical cut-off concentration is usually only 1 μg per mL.

A large group of enrichment methods applies various capturing reagents, such as antibodies, specific ligands, combinations of antibodies and ligands and peptide ligand libraries [25]. ProteoMiner is another equalizing method, which is used for enrichment of LAPs by applying a comprehensive bead-based spectrum of peptide ligands, which enrich LAPs and dilute high abundance proteins. Several studies have reported the effective elimination of high abundance proteins in human serum [19], synovial fluid [18] and urine [26] and Pseudomonas membrane protein extracts [20]. Abundance equalization has also been applied for analysis of the soluble fraction of skeletal muscle [27]. In addition, immunodepletion with heavy chain IgG antibodies combined with lectin affinity chromatography has been used to enrich glycoproteins in human serum [28].

Immunoaffinity-based partitioning of high abundant proteins allows the characterization of LAPs [29]. The method applies avian polyclonal IgY antibodies to perform effective partitioning of protein fractions. Sigma provides Seppro IgY14 column system (SEP010-1KT), which is designed to eliminate the main 14 high abundant proteins. In addition, there is the SuperMix column, which can be used to separate moderately abundant proteins from LAPs.

The enrichment of LAPs may also be achieved by heparin chromatography, which is characterized by the reversible adsorption of peptides by immobilized heparins. Heparin is well known for having a strong protein binding ability and can distinguish and enrich LAPs with small dissimilarities in glycosylation characteristics. In particular, subcellular fractionation followed by heparin chromatography has been used to enrich secreted rat brain LAPs [10]. In addition, fibroblast growth factors isolated from mouse endotheliocytes have been fractionated using heparin chromatography [11]. Furthermore, heparin chromatography has been combined with protein G sepharose in order to remove highly abundant serum proteins [30].

Mass spectrometry, Western blotting, ELISA and nuclear magnetic resonance have been used to detect LAPs (Table 2).

Mass spectrometry with signal augmentation is used to detect LAPs. Single-cell proteomics with mass spectrometry is an actively studied topic [36, 37]. A recent study has described the binding of LAPs to gold nanoparticles and substrates (Fig. 2) [1]. Using the detection of epithelial cell adhesion molecule (EpCAM) as an example, the study has shown that a signal can be obtained even from a very low amount of EpCAM with the detection limit of 0.2 zM or approximately 120 molecules at the concentration 100 aM.

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Figure 2. Application of EDTA-functionalized magnetic nanoparticles for protein fractionation prior to liquid chromatography-mass spectrometry analysis. EDTA-functionalized magnetic nanoparticles have been used to deplete proteins of high abundance in urine samples in order to improve the characterization of low abundance proteins by mass spectrometry [2].

Moreover, positive selection of target biomarkers has been shown to improve the analysis. Affinity capture reagents can improve the yield of LAPs with the concentration range 0.1-10 pg/mL [38]. The spectrum of proteins has been analyzed at a concentrations of 35 mM. The results of this method are highly tolerant of the diversity of structure and size. The authors have shown that the target molecule can be analyzed even if the oligomer constitutes just 5% of the total mixture. Moreover, this technique does not require isotopes.

Furthermore, a new method, which combined chromatography and mass spectrometry has recently been developed [39]. This method has applied iTRAQ four-plex labeling, which has allowed identification of 5300 proteins. In comparison to a label-free method, the iTRAQ four-plex labeling-based technique characterized fivefold more proteins and showed up to a nine-fold enhancement of detection throughput.

Western blotting can be used to characterize small size proteins, 5–10 μg total mass (1–3 μg total protein) [40]. The combination of Western blot with immunoprecipitation has been shown to significantly improve the identification and analysis of LAPs [31]. Furthermore, the characterization of LAPs by WB can be significantly improved by several optimization techniques. The approaches for LAP recovery include selection of appropriate lysis buffer, protein precipitation, optimization of gel electrophoresis, concentration of primary antibody and signal detection [41]. In addition, AdvanBlock™-chemi, a new blocking reagent for chemiluminescent Western blotting, has recently been used to enhance positive signal and reduce nonspecific binding [32]. This reagent allows steady detection of LAPs using 0.03-0.5 μg (Jurkat cells) or 0.2-5 μg (HeLa cells) of the whole cell lysates. The AdvanBlock™-chemi-based method has been shown to reduce nonspecific background and increase the positive specific signal by stabilizing of the antigen-antibody binding.

Since a standard ELISA is not sensitive enough to characterize LAPs, another sensitive method for LAP detection, combining ELISA with a proximity ligation assay (PLA), has been described by Tong et al [33]. Using this method, named ELISA-PLA, the specificity was enhanced by synchronous detection of target molecules via numerous probes. In addition, rolling circle amplification was applied for mending the sensitivity. Previous studies have shown that LAPs can be detected in the pg/mL ranges by ELISA [34]. Simoa technology, a variation of sandwich ELISA, can detect a target at 10^-19 M level [42]. A similar assay, Proximity Extension Assay, can alos detect low levels of proteins among the bodily fluids [43]. Arboleda-Velasquez JF et al measured plasma NfL levels with an in-house simoa assay [44], so did YT Quiroz et al [45]. Holth JK et al used Simoa Human Neurology 4-Plex “A” assay and Alpha-Synuclein Discovery Kit (both from Quanterix) to measure human cerebrospinal fluid NfL, GFAP, and synuclein levels to investigate the effect of sleep deprivation on tau and amyloid beta accumulation [46]. Meso Scale Diagnostics technology can also achieve similar sensitivity. Poorbaugh J et al compared MSD S-PLEX and Quanterix SiMoA methodologies for the detection of IL-21 in human serum and plasma, and found the Limit of Detection [LOD] for both assays was approximately 1.0 fg/mL [47].

Another effective technique to characterize LAPs is nuclear magnetic resonance (NMR) with high resolution. In particular, NMR has been applied for analysis of amyloid-β protein, which is a specific marker of Alzheimer’s disease [35]. Magic angle spinning recoupling ¹H-¹H NMR method has allowed sub-molecular evaluation of low abundance amyloid Aβ1-40 peptide. This method in its solution- and solid-state variations allowed structural characterization of the intermediate oligomers (5-10 nm) and may be used for analysis of about only 6 μg of oligomers.

The subcellular localization of LAP can be facilitated through the "spaghetti monster" tagging of the LAP [48].

Proteins with low abundance are usually difficult to analyze without the optimization of standard methods, such as mass spectrometry or Western blotting. Therefore, most of these methods require additional signal amplification steps. Depletion of highly abundant proteins by various methods, such as collagenase, isopropanol, polyethylene glycol, protamine sulfate or nanoparticles, can be performed to enhance the specific signals for LAPs. With regard to current methods of LAP detection, which include mass spectrometry, Western blotting, ELISA and NMR, a combination with various enrichment approaches significantly improves the characterization of LAPs.