Peptide Mass Fingerprinting Research Articles

Detection of Frozen-Thawed Duck Fatty Liver by MALDI-TOF Mass Spectrometry: A Chemometrics Study.

The marketing of poultry livers is only authorized as fresh, frozen, or deep-frozen. The higher consumer demand for these products for a short period of time may lead to the marketing of frozen–thawed poultry livers: this constitutes fraud. The aim of this study was to design a method for distinguishing frozen–thawed livers from fresh livers. For this, the spectral fingerprint of liver proteins was acquired using Matrix-Assisted Laser Dissociation Ionization-Time-Of-Flight mass spectrometry. The spectra were analyzed using the chemometrics approach. First, principal component analysis studied the expected variability of commercial conditions before and after freezing–thawing. Then, the discriminant power of spectral fingerprint of liver proteins was assessed using supervised model generation. The combined approach of mass spectrometry and chemometrics successfully described the evolution of protein profile during storage time, before and after freezing-thawing, and successfully discriminated the fresh and frozen–thawed livers. These results are promising in terms of fraud detection, providing an opportunity for implementation of a reference method for agencies to fight fraud.

Automated annotation and visualisation of high-resolution spatial proteomic mass spectrometry imaging data using HIT-MAP

Spatial proteomics has the potential to significantly advance our understanding of biology, physiology and medicine. Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a powerful tool in the spatial proteomics field, enabling direct detection and registration of protein abundance and distribution across tissues. MALDI-MSI preserves spatial distribution and histology allowing unbiased analysis of complex, heterogeneous tissues. However, MALDI-MSI faces the challenge of simultaneous peptide quantification and identification. To overcome this, we develop and validate HIT-MAP (High-resolution Informatics Toolbox in MALDI-MSI Proteomics), an open-source bioinformatics workflow using peptide mass fingerprint analysis and a dual scoring system to computationally assign peptide and protein annotations to high mass resolution MSI datasets and generate customisable spatial distribution maps. HIT-MAP will be a valuable resource for the spatial proteomics community for analysing newly generated and retrospective datasets, enabling robust peptide and protein annotation and visualisation in a wide array of normal and disease contexts.

Pleiotropic effects of ActVI-ORFA as an unusual regulatory factor identified in the biosynthetic pathway of actinorhodin in Streptomyces coelicolor

Regulatory networks play critical roles in controlling the the biosynthesis of natural products in Streptomyces. ActVI-ORFA, a regulatory factor encoded by the actinorhodin biosynthetic gene cluster (act cluster), positively controls the production of actinorhodin (ACT) in Streptomyces coelicolor, although its regulatory mechanism remains obscure. This study aimed to identify the regulatory targets of ActVI-ORFA. Deletion of ActVI-ORFA caused the differential expression of hundreds of proteins, as determined by two-dimensional electrophoresis and peptide mass fingerprinting analysis. qRT-PCR analysis of some genes encoding these differentially expressed proteins, including act genes and non-act genes, confirmed that ActVI-ORFA could control their transcriptional levels. In an electrophoretic mobility shift assay with a promoter region of a target gene located in the act cluster, no binding was detected, consistent with the lack of a recognizable DNA-binding domain in ActVI-ORFA. Overall, our findings suggest that ActVI-ORFA is a pleiotropic regulatory factor that controls multiple physiological pathways, including secondary metabolite production, probably via an indirect mode.

A magainin-2 like bacteriocin BpSl14 with anticancer action from fish gut Bacillus safensis SDG14

Bacteriocins are gaining utmost importance in antimicrobial and chemotherapy due to their diverse structure and activity. This study centres on magainin-2 like bacteriocin with anticancer action, produced by Bacillus safensis strain SDG14 isolated from gut of marine fish Sardinella longiceps. The purified bacteriocin designated as BpSl14 was thermostable and pH tolerant. The molecular weight of BpS114 was estimated to be 6061.2 Da using MALDI-ToF MS. The partial primary sequence was elucidated by peptide mass fingerprinting using MALDI MS/MS. The tertiary structure of the partial sequence was similar to that of two magainin-2 α-helices joined together by extended indolicidin. The BpSl14 protein inhibited the cells of lung carcinoma, one of the deadliest cancers. Docking studies conducted with DR5 and TGF-β, two of the most prominent apoptotic receptors in adenocarcinoma, also proved the anti-apoptotic action of BpSl14.

The last caravans in antiquity: Camel remains from Caričin Grad (Justiniana Prima)

Camels played an important role in caravan traffic and long-distance communication during the early Byzantine period as indicated by new findings from Caričin Grad. Excavations at the site revealed the remains of an important early Byzantine city, which has been identified with Justiniana Prima founded in the 530 s by Justinian I (527–565). The city, created ex nihilo, in a rural area in the north-west of the province of Dacia Mediterranea, represents a distinctive example of late urbanisation. Given the existence of the city for only 80 years and the fact that the nearby locality remained uninhabited until modern times, the site has yielded well-preserved monuments and remains of material culture. They allow for a detailed study of the urbanism, architecture, material culture and other aspects of daily life from the second quarter of the 6th century to the beginning of the 7th century. Here we investigate the camel remains discovered in five locations in Caričin Grad. Morphometric and proteomic analyses, including both collagen peptide mass fingerprint analysis (ZooMS) as well as in-depth sequencing, revealed that the majority originate from hybrids of the dromedary and Bactrian camels. Supported by written sources from the Early Byzantine period, it appears as though the use of the Caričin Grad camels was primarily for civilian purposes. These results highlight the complementarity of morphometric and proteomic approaches and their value in better understanding the impact of the early Byzantine economy.

Distinguishing African bovids using Zooarchaeology by Mass Spectrometry (ZooMS): New peptide markers and insights into Iron Age economies in Zambia.

Assessing past foodways, subsistence strategies, and environments depends on the accurate identification of animals in the archaeological record. The high rates of fragmentation and often poor preservation of animal bones at many archaeological sites across sub-Saharan Africa have rendered archaeofaunal specimens unidentifiable beyond broad categories, such as “large mammal” or “medium bovid”. Identification of archaeofaunal specimens through Zooarchaeology by Mass Spectrometry (ZooMS), or peptide mass fingerprinting of bone collagen, offers an avenue for identification of morphologically ambiguous or unidentifiable bone fragments from such assemblages. However, application of ZooMS analysis has been hindered by a lack of complete reference peptide markers for African taxa, particularly bovids. Here we present the complete set of confirmed ZooMS peptide markers for members of all African bovid tribes. We also identify two novel peptide markers that can be used to further distinguish between bovid groups. We demonstrate that nearly all African bovid subfamilies are distinguishable using ZooMS methods, and some differences exist between tribes or sub-tribes, as is the case for Bovina (cattle) vs. Bubalina (African buffalo) within the subfamily Bovinae. We use ZooMS analysis to identify specimens from extremely fragmented faunal assemblages from six Late Holocene archaeological sites in Zambia. ZooMS-based identifications reveal greater taxonomic richness than analyses based solely on morphology, and these new identifications illuminate Iron Age subsistence economies c. 2200–500 cal BP. While the Iron Age in Zambia is associated with the transition from hunting and foraging to the development of farming and herding, our results demonstrate the continued reliance on wild bovids among Iron Age communities in central and southwestern Zambia Iron Age and herding focused primarily on cattle. We also outline further potential applications of ZooMS in African archaeology.

Isolation, characterization of galactose-specific lectin from Odoiporus longicollis and its antibacterial and anticancer activities

Lectins are renowned hemagglutinins and multivalent proteins with a well known quality for sugar-binding specificity that participate significantly in invertebrate defense functions. Studies on biological activity of lectin from coleopteran insect are very scarce. In this study, lectin from the hemolymph in the grub of banana pest, Odoiporus longicollis was subjected to purification, biochemical and functional characterizations. The lectin was purified by PEG precipitation and ion-exchange chromatography using Q-Sepharose as a matrix. The purified lectin showed hemagglutination activity against rat erythrocytes, heat-labile, cation independent and insensitive to EDTA. Further, the carbohydrate affinity of this lectin was found with mannitol, adonitol, L-arabinose, L-rhamnose, D-galactose and sorbitol. The native form of purified lectin was calculated as 360 kDa by FPLC system. Denatured gel electrophoresis of the purified lectin consisted of five distinct polypeptides with molecular weights approximately 160, 60, 52, 40 and 38 kDa, respectively. The amino acid sequences obtained through peptide mass fingerprinting analysis exhibited homologies to the known conserved regions of galactose binding lectins. Further, the purified lectin exhibited bacterial inhibition with LPS from Serratia marcescens. In addition, isolated lectin also exerted bacterial agglutination, antibacterial and anti-proliferative activity against Mycobacterium smegmatis, Bacillus pumilus and Neuro 2a cell line, respectively.

Streptococcus suis serotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Streptococcus suis, particularly S. suis serotype 2 (SS2), is an important zoonotic pathogen causing meningitis in humans worldwide. Although the proper classification of the causative and pathogenic serotype is salutary for the clinical diagnosis, cross-reactions leading to the indistinguishability of serotypes by the current serotyping methods are significant limitations. In the present study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of extracted peptides was developed to improve the classification of serotype of S. suis. The peptide mass fingerprint (PMFs) database of S. suis was generated from the whole-cell peptides of 32 reference strains of S. suis isolates obtained from pigs. Thirty-two human S. suis isolates from clinical cases in Thailand were used to validate this alternative serotyping method in direct comparison to the multiplex (m)PCR approach. All reference strains, representing 32 serotypes of S. suis, exhibited their individual PMFs patterns, thus allowing differentiation from one another. Highly pathogenic SS2 and SS14 were clearly differentiated from the otherwise serologically closely related SS1/2 and SS1, respectively. The developed MALDI-TOF-MS serotyping method correctly classified the serotype in 68.8% (22/32) of the same serotype isolates generated from the PMFs database; while the validity for the clinical human isolates was 62.5% (20/32). The agreement between the MALDI-TOF-MS and mPCR serotyping was moderate with a Kappa score of 0.522, considering that mPCR could correctly serotype up to 75%. The present study demonstrated that PMFs from the developed MALDI-TOF-MS-based method could successfully discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively. Moreover, this serotyping method distinguished pathogenic SS6, and so is an alternative approach of choice to rapidly and reliably serotype clinically pathogenic S. suis isolates.

Quantification of Local Electric Field Changes at the Active Site of Cytochrome c Oxidase by Fourier Transform Infrared Spectroelectrochemical Titrations.

Cytochrome c oxidase (CcO) is a transmembrane protein complex that reduces molecular oxygen to water while translocating protons across the mitochondrial membrane. Changes in the redox states of its cofactors trigger both O2 reduction and vectorial proton transfer, which includes a proton-loading site, yet unidentified. In this work, we exploited carbon monoxide (CO) as a vibrational Stark effect (VSE) probe at the binuclear center of CcO from Rhodobacter sphaeroides. The CO stretching frequency was monitored as a function of the electrical potential, using Fourier transform infrared (FTIR) absorption spectroelectrochemistry. We observed three different redox states (R4CO, R2CO, and O), determined their midpoint potential, and compared the resulting electric field to electrostatic calculations. A change in the local electric field strength of +2.9 MV/cm was derived, which was induced by the redox transition from R4CO to R2CO. We performed potential jump experiments to accumulate the R2CO and R4CO species and studied the FTIR difference spectra in the protein fingerprint region. The comparison of the experimental and computational results reveals that the key glutamic acid residue E286 is protonated in the observed states, and that its hydrogen-bonding environment is disturbed upon the redox transition of heme a3. Our experiments also suggest propionate A of heme a3 changing its protonation state in concert with the redox state of a second cofactor, heme a. This supports the role of propionic acid side chains as part of the proton-loading site.

Organization of the Structural Protein Region of La Jolla Virus Isolated from the Invasive Pest Insect Drosophila suzukii.

Drosophila suzukii (Ds) is an invasive pest insect that infests ripening fruit, causing severe economic losses. Control measures based on chemical pesticides are inefficient and undesirable, so biological alternatives have been considered, including native Ds viruses. We previously isolated a strain of La Jolla virus (LJV-Ds-OS20) from Ds in Germany as a candidate biopesticide. Here we characterized the new strain in detail, focusing on the processing of its capsid proteins. We tested LJV growth during Ds development to optimize virus production, and established a laboratory production system using adult flies. This system was suitable for the preparation of virions for detailed analysis. The LJV-Ds-OS20 isolate was cloned by limiting dilution and the complete nucleotide sequence was determined as a basis for protein analysis. The terminal segments of the virus genome were completed by RACE-PCR. LJV virions were also purified by CsCl gradient centrifugation and analyzed by SDS-PAGE and electron microscopy. The capsid proteins of purified LJV virions were resolved by two-dimensional SDS-PAGE for N-terminal sequencing and peptide mass fingerprinting. The N-terminal sequences of VP1 and VP2, together with MS data representing several capsid proteins, allowed us to develop a model for the organization of the LJV structural protein region. This may facilitate the development of new viral strains as biopesticides.

Iron Age hunting and herding in coastal eastern Africa: ZooMS identification of domesticates and wild bovids at Panga ya Saidi, Kenya

The morphological differentiation of African bovids in highly fragmented zooarchaeological assemblages is a major hindrance to reconstructing the nature and spread of pastoralism in sub-Saharan Africa. Here we employ collagen peptide mass fingerprinting, known as Zooarchaeology by Mass Spectrometry (ZooMS), coupled with recently published African ZooMS reference datasets, to identify domesticates and wild bovids in Iron Age assemblages at the cave site of Panga ya Saidi in southeast Kenya. Through ZooMS we have identified all three major African livestock—sheep (Ovis aries), goat (Capra hircus) and cattle (Bos taurus)—at the site for the first time. The results provide critical evidence for the use of domesticates by resident foraging populations during the Iron Age, the period associated with the arrival of food production in coastal Kenya. ZooMS results show that livestock at Panga ya Saidi form a minor component of the assemblage compared to wild bovids, demonstrating the persistence of hunting and the secondary role of acquiring livestock in hunter-gatherer foodways during the introduction of agro-pastoralism. This study sheds new light on the establishment of food production in coastal eastern Africa, particularly the role of interactions between hunter-gatherers and neighbouring agro-pastoral groups in what was a protracted regional transition to farming.

Construction and Application of MALDI-TOF Mass Spectrometry for the Detection of Haemophilus parasuis.

To construct a protein fingerprint database of Haemophilus parasuis (H. parasuis), thus improving its clinical diagnosis efficiency. A total of 15 H. parasuis standard strains were collected to establish a protein fingerprint database of H. parasuis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the effects of different culture media and culture time on the quality and identification results of the protein fingerprint were investigated. The results showed that tryptone soy agar (TSA) and tryptone soy broth (TSB) media and different incubation times had no significant effect on the characteristic peaks of the protein profiles. In addition, 18 clinical isolates were used to compare the identification results of the self-built protein fingerprint database, PCR detection, and basic database. Only one strain was identified in the original VITEK-MS system database, while the self-made protein fingerprint database of H. parasuis was 100% accurate for the detection of 18 clinical isolate strains. The protein fingerprint database of H. parasuis built by our laboratory is suitable for rapid clinical diagnosis of H. parasuis, due to its high accuracy, efficiency, and strong specificity.

Tubulin Beta 2C Chain (TBB2C), a Potential Marker of Ovarian Cancer, an Insight from Ovarian Cancer Proteome Profile.

Ovarian cancer (OC) is the most lethal among female reproductive system malignancies. Depending upon the stage at presentation, the five year survival ratio varies from ∼92 to ∼30%. The role of biomarkers in early cancer diagnosis, including OC, is well understood. In our previous study, through an initial screening, we have analyzed eleven proteins that exhibited differential expression in OC using two-dimensional gel electrophoresis (2D-GE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometric (MALDI-TOF MS) analysis. In continuation of our previous study, the present work describes analysis of twenty more proteins that showed aberrant expression in OC. Among these, six showed consistent significant deregulation in the OC false discovery rate [FDR ≤ 0.05]. Upon MS analysis, they were identified as vimentin, tubulin beta 2C chain, tubulin alpha 1C chain, actin cytoplasmic 2, apolipoprotein A-I, and collagen alpha 2(VI) chain [peptide mass fingerprint (PMF) score ≥ 79]. One of the differentially regulated proteins, tubulin beta 2C chain, was found to be significantly (fold change, 2.5) enhanced in OC. Verification by western blot and enzyme-linked immunosorbent assay (ELISA) demonstrated that the tubulin beta 2C chain may serve as a valuable marker for OC (ANOVA p < 0.0001). The assessment of the likely association of TBB2C with OC in a larger population will not only help in developing clinically useful biomarkers in the future but also improve our understanding of the progression of OC disease.

Evidence for early dispersal of domestic sheep into Central Asia.

The development and dispersal of agropastoralism transformed the cultural and ecological landscapes of the Old World, but little is known about when or how this process first impacted Central Asia. Here, we present archaeological and biomolecular evidence from Obishir V in southern Kyrgyzstan, establishing the presence of domesticated sheep by ca. 6,000 BCE. Zooarchaeological and collagen peptide mass fingerprinting show exploitation of Ovis and Capra, while cementum analysis of intact teeth implicates possible pastoral slaughter during the fall season. Most significantly, ancient DNA reveals these directly dated specimens as the domestic O. aries, within the genetic diversity of domesticated sheep lineages. Together, these results provide the earliest evidence for the use of livestock in the mountains of the Ferghana Valley, predating previous evidence by 3,000 years and suggesting that domestic animal economies reached the mountains of interior Central Asia far earlier than previously recognized.

The pyruvate:ferredoxin oxidoreductase of the thermophilic acetogen, Thermoanaerobacter kivui.

Pyruvate:ferredoxin oxidoreductase (PFOR) is a key enzyme in bacterial anaerobic metabolism. Since a low‐potential ferredoxin (Fd2−) is used as electron carrier, PFOR allows for hydrogen evolution during heterotrophic growth as well as pyruvate synthesis during lithoautotrophic growth. The thermophilic acetogenic model bacterium Thermoanaerobacter kivui can use both modes of lifestyle, but the nature of the PFOR in this organism was previously unestablished. Here, we have isolated PFOR to apparent homogeneity from cells grown on glucose. Peptide mass fingerprinting revealed that it is encoded by pfor1. PFOR uses pyruvate as an electron donor and methylene blue (1.8 U·mg−1) and ferredoxin (Fd; 27.2 U·mg−1) as electron acceptors, and the reaction is dependent on thiamine pyrophosphate, pyruvate, coenzyme A, and Fd. The pH and temperature optima were 7.5 and 66 °C, respectively. We detected 13.6 mol of iron·mol of protein−1, consistent with the presence of three predicted [4Fe–4S] clusters. The ability to provide reduced Fd makes PFOR an interesting auxiliary enzyme for enzyme assays. To simplify and speed up the purification procedure, we established a protocol for homologous protein production in T. kivui. Therefore, pfor1 was cloned and expressed in T. kivui and the encoded protein containing a genetically engineered His‐tag was purified in only two steps to apparent homogeneity. The homologously produced PFOR1 had the same properties as the enzyme from T. kivui. The enzyme can be used as auxiliary enzyme in enzymatic assays that require reduced Fd as electron donor, such as electron‐bifurcating enzymes, to keep a constant level of reduced Fd.

Streptococcus mutans detection on mother-child pairs using matrix-assisted laser desorption ionization – time of flight mass spectrometry and polymerase chain reaction

Background: Streptococcus mutans (S. mutans) bacteria mainly cause dental caries in children. These bacteria are not considered oral indigenous bacteria since they are transmitted from people around children during their deciduous teeth eruption. The detection of these bacteria can be used for dental caries prevention in children. Purpose: To determine the strain and serotype of S. mutans by using matrix assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) and polymerase chain reaction (PCR) on dental plaque samples taken from mother-child pairs. Methods: Sixteen dental plaque samples of mother-child pairs were cultured on brain heart infusion broth (BHIB) and mitis salivarius bacitracin (MSB) media until S. mutans colony isolates were obtained. Next, the isolates of S. mutans colony were introduced into the target plates of MALDI-TOF MS, and then ionized to become peptide mass fingerprint (PMF). Afterwards, the colony isolates were detected by database software. The detected S. mutans DNA then was extracted by using conventional 727 bp PCR (serotype C). Results: Six strains of S. mutans were detected by MALDI-TOF MS method. Five samples were classified into UA159, two samples were 3SN1, two samples were NFSM1, two samples were 11A1, two samples were U138, two samples were 4SM1, and one sample was classified into another bacterium. Five out of 16 samples were detected by PCR as serotype C (UA159). Conclusion: Six strains of S. mutans were detected, namely UA159, 3SN1, NFSM1, 11A1, U138, and 4SM1, one of them (UA159) was detected as serotype C.

Scratching the surface: the use of sheepskin parchment to deter textual erasure in early modern legal deeds

Historic legal deeds are one of the most abundant resources in British archives, but also one of the most neglected. Despite the millions that survive, we know remarkably little about their manufacture, including the species of animal on which they were written. Here we present the species identification of 645 sixteenth–twentieth century skins via peptide mass fingerprinting (ZooMS), demonstrating the preferential use of sheepskin parchment. We argue that alongside their abundance and low cost, the use of sheepskins over those of other species was motivated by the increased visibility of fraudulent text erasure and modification afforded by the unique structure of their skin.

Palaeoproteomics confirm earliest domesticated sheep in southern Africa ca. 2000 BP

We used palaeoproteomics and peptide mass fingerprinting to obtain secure species identifications of key specimens of early domesticated fauna from South Africa, dating to ca. 2000 BP. It can be difficult to distinguish fragmentary remains of early domesticates (sheep) from similar-sized local wild bovids (grey duiker, grey rhebok, springbok—southern Africa lacks wild sheep) based on morphology alone. Our analysis revealed a Zooarchaeology by Mass Spectrometry (ZooMS) marker (m/z 1532) present in wild bovids and we demonstrate through LC–MS/MS that it is capable of discriminating between wild bovids and caprine domesticates. We confirm that the Spoegrivier specimen dated to 2105 ± 65 BP is indeed a sheep. This is the earliest directly dated evidence of domesticated animals in southern Africa. As well as the traditional method of analysing bone fragments, we show the utility of minimally destructive sampling methods such as PVC eraser and polishing films for successful ZooMS identification. We also show that collagen extracted more than 25 years ago for the purpose of radiocarbon dating can yield successful ZooMS identification. Our study demonstrates the importance of developing appropriate regional frameworks of comparison for future research using ZooMS as a method of biomolecular species identification.

Draft genome of the glucose tolerant β-glucosidase producing rare Aspergillus unguis reveals complete cellulolytic machinery with multiple beta-glucosidase genes

Draft genome sequence of the glucose tolerant beta glucosidase (GT-BGL) producing rare fungus Aspergillus unguis NII 08,123 was generated through Next Generation Sequencing (NGS). The genome size of the fungus was estimated to be 37.1 Mb. A total of 3116 contigs were assembled using SPades, and 15,161 proteins were predicted using AUGUSTUS 3.1. Among them, 13,850 proteins were annotated using UniProt. Distribution of CAZyme genes specifically those encoding lignocellulose degrading enzymes were analyzed and compared with those from the industrial cellulase producer Trichoderma reesei in view of the huge differences in detectable enzyme activities between the fungi, despite the ability of A. unguis to grow on lignocellulose as sole carbon source. Full length gene sequence of the inducible GT-BGL could be identified through tracing back from peptide mass fingerprint. A total of 403 CAZymes were predicted from the genome, which includes 232 glycoside hydrolases (GHs), 12 carbohydrate esterases (CEs), 109 glycosyl transferases (GTs), 15 polysaccharide lyases (PLs), and 35 genes with auxiliary activities (AAs). The high level of zinc finger motif containing transcription factors could possibly hint a tight regulation of the cellulolytic machinery, which may also explain the low cellulase activities even when a complete repertoire of cellulase degrading enzyme genes are present in the fungus.

Novel Method for the Isolation of Proteins and Small Target Molecules from Biological and Aqueous Media by Salt-Assisted Phase Transformation of Their PEGylated Recognition Counterparts.

An efficient and simple method for the application of PEGylated affinity ligands in precipitative isolation of protein target molecules (TMs) from a biological fluid such as blood serum or small target molecules from an aqueous medium is presented for the first time. This approach is based on the high binding specificity of PEGylated recognition molecules (PEG-RMs) to their TMs and the unique physicochemical properties of PEG that result in their salt-assisted phase transformation. Addition of PEG-RM to blood serum results in the formation of an RM-specific macromolecular complex (PEG-RM + TM → PEG-RM.TM) that undergoes facile salt-assisted phase transformation to a separable semisolid with ammonium sulfate. PEG-RM.TM is then dissociated into its components by pH reduction or an increase of ionic strength (PEG-RM.TM → PEG-RM + TM). PEG-RM is salted out to afford pure TM in solution. The same phenomenon is observed when RM or TM are small molecules. The general applicability of the method was validated by PEGylation of two proteins (protein A, sheep antihuman IgG) and a small molecule (salicylic acid) used as model RMs for the isolation of Igs, IgG, and serum albumin from blood serum. The isolated protein TMs were shown to be pure and aggregate-free by gel electrophoresis and dynamic light scattering (DLS). IgG isolated by this method was further characterized by peptide mass fingerprinting. PEGylated protein A was used to demonstrate the recyclability and scale-up potential of PEG-RM. IgG isolated by this method from blood serum of a hepatitis C-vaccinated individual was tested for its binding to sheep antihuman IgG by UV spectroscopy, and its bioactivity was ascertained by comparison of its enzyme-linked immunosorbent assay (ELISA) result to that of a blood sample from the same individual. Reciprocity of RM and TM was ascertained using PEGylated salicylic acid to obtain pure serum albumin, and PEGylated serum albumin was utilized for near-exclusive isolation of one drug from an aqueous equimolar mixture of three drugs (salicylic acid, 91%; capecitabine, 6%; and deferiprone, 3%). Advantages of this approach, including target specificity and general applicability and celerity, over other affinity methods for the isolation of proteins are discussed at a molecular level.