Standard Flow Multiplexed Proteomics (SFloMPro)—An Accessible Alternative to NanoFlow Based Shotgun Proteomics

Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid “standard flow” HPLC gradient LC-MS/MS. Standard Flow Multiplexed Proteomics (SFloMPro) enables high coverage quantitative proteomics of up to 16 mammalian samples in about 24 h. In this study, we compare NanoLC and SFloMPro analysis of fractionated samples. Our results demonstrate that comparable data is obtained by injecting 20 µg of labeled peptides per fraction with SFloMPro, compared to 1 µg per fraction with NanoLC. We conclude that, for experiments where protein concentration is not strictly limited, SFloMPro is a competitive approach to traditional NanoLC workflows with improved up-time, reliability and at a lower relative cost per sample.

Proteomic analysis of extracellular vesicles enriched serum associated with future ischemic stroke

Identifying new biomarkers beyond the established risk factors that make it possible to predict and prevent ischemic stroke has great significance. Extracellular vesicles are powerful cell‒cell messengers, containing disease-specific biomolecules, which makes them powerful diagnostic candidates. Therefore, this study aimed to identify proteins derived from extracellular vesicles enriched serum related to future ischemic stroke events, using a proteomic method. Of Japanese subjects who voluntarily participated in health checkups at our institute a number of times, 10 subjects (6 males and 4 females, age: 64.2 ± 3.9 years) who developed symptomatic ischemic stroke (7.3 ± 4.4 years’ follow-up) and 10 age‒sex matched controls without brain lesions (6.7 ± 2.8 years’ follow-up) were investigated. Extracellular vesicles enriched fractions were derived from serum collected at the baseline visit. Differentially expressed proteins were evaluated using isobaric tagging for relative and absolute protein quantification (iTRAQ)-based proteomic analysis. Of the 29 proteins identified, alpha-2-macroglobulin, complement C1q subcomponent subunit B, complement C1r subcomponent, and histidine-rich glycoprotein were significantly upregulated (2.21-, 2.15-, 2.24-, and 2.16-fold, respectively) in subjects with future ischemic stroke, as compared with controls. Our study supports the concept of serum-derived extracellular vesicles enriched fractions as biomarkers for new-onset stroke. These proteins may be useful for prediction or for targeted therapy.

Aliquoting of isobaric labeling reagents for low concentration and single cell proteomics samples

The introduction of isobaric tagging reagents enabled more accurate, high-throughput quantitative proteomics by enabling samples to be multiplexed. One drawback of these workflows is the relative expense of the proprietary chemical reagents, which is often only second to the expense of the instruments themselves. These highly reactive chemical tags are only commercially available in relatively large aliquots compared to the typical amounts of peptides analyzed in proteomic workflows today. Excess reagents are typically disposed of following labeling of small batches or within a few weeks of opening. We present a simple procedure to aliquot commercial isobaric tagging reagents and demonstrate the successful and high efficiency labeling of multiple samples over a period of six months. The samples presented herein were selected as the most diverse samples labeled by prepared aliquots from a single labeling reagent kit. We observe comparable labeling efficiency from 100 microgram to 100 picograms of peptide when labeling samples from both human digest standards, cancer cell lines prepared in-house and from cells directly obtained from organ donations, despite differences in cell type, lysis and digestion. No labeling experiment of whole human proteomics samples achieved less than 92% labeling efficiency over this period. When preparing phosphoproteomic samples from a cancer cell line digest at approximately 6 months from the date of the aliquoting procedure, we observed a decrease in labeling efficiency to approximately 86%, indicating we are approaching the end of the useful lifetime of these prepared aliquots. Over this period, we have effectively reduced the reagent costs of each labeling experiment to less than 10% of the predicted costs when following the manufacturer instructions for use and disposal. While aliquoting of reagents can be performed by hand, we provide a complete template for automatic aliquoting using an affordable liquid handling robot, including plans for 3D printing of parts we have found useful for streamlining this procedure.

Automation of On-Resin Enrichment of S-Nitrosylated Proteins for Oxidized Cysteine-Selective cPILOT

S-Nitrosylation (SNO) is a cysteine post-translational modification that increases with normal aging and is present in Alzheimer’s disease and other aging-related illnesses. Detection of SNO-modified proteins can be challenging; however, we previously developed a robust quantitative proteomics approach termed “Oxidized Cysteine-Selective combined precursor isobaric labeling and isobaric tagging (OxcyscPILOT)” that allows for detection of endogenous SNO-modified proteins. OxcyscPILOT involves enrichment of SNO-modified proteins using a thiol-based resin. This enrichment is performed manually, and wash stages with the resin require numerous steps and buffer reagents. The goal of this study is to transfer the manual protocol to an automated liquid handler system in order to reduce wash steps, increase sample throughput, and minimize experimental error. In order to accomplish this, we evaluated the Biomek i7 liquid handler automated workstation and a Positive Pressure ALP (PPA) apparatus as a means to conduct automated on resin enrichment. Our findings provide starting pressure conditions for the use of PPA in an automated OxcyscPILOT proteomics workflow that could be transferred to other robotic liquid handling systems.

Stability of amino acids and related amines in human serum under different preprocessing and pre-storage conditions based on iTRAQ®-LC-MS/MS

ABSTRACTAmino acid analysis or metabonomics requires large-scale sample collection, which makes sample storage a critical consideration. However, functional amino acids are often neglected in metabolite stability studies because of the difficulty in detecting and accurately quantifying them with most analysis methods. Here, we investigated the stability of amino acids and related amines in human serum following different preprocessing and pre-storage procedures. Serum samples were collected and subjected to three storage conditions; cold storage (4°C), room temperature storage (22°C), and freezing (−80°C). The concentration of amino acids and related amines were quantified using iTRAQ®-LC-MS/MS with isobaric tagging reagents. Approximately 54.84%, 58.06%, and 48.39% of detectable and target analytes were altered at the 4°C condition, 22°C condition, and when subjected to freeze-thaw cycles, respectively. Some amino acids which are unstable and relatively stable were found. Our study provides detailed amino acid profiles in human serum and suggests pre-treatment measures that could be taken to improve stability.

The Proteomics and Fibromyalgia: A Perspective on the Study of the Inflammatory Response in Fibromyalgia

Fibromyalgia (FM) is a chronic, non-degenerative disease characterized by widespread and sustained pain, sleep disturbances, physical exhaustion, and cognitive difficulties. The FM pathophysiology has not been completely clarified, and several theories have been postulated, among which is the dysregulation of the inflammatory response as a mediator of the painful phenomenon. In addition, it has been reported that FM patients present a rise of IL-6 and IL-8 serum levels; this fact has clinical relevance since these inflammatory molecules induce symptoms such as pain, fatigue, hyperalgesia, and allodynia. Additionally, some studies have been carried out on the participation of leukocytes in the physiopathology of fibromyalgia; The evidence suggests that mast cells are the most relevant leukocytes in the pathophysiology of FM since they promote the release of pro-inflammatory cytokines in response to stimuli such as substance P or corticotropin-releasing hormone, which were elevated in patients. The number of FM patients is increasing year after year around the world. For this reason, it is essential to study the proteins involved in the inflammatory response in fibromyalgia. Proteomic analysis techniques such as tandem mass tag (TMT) with isobaric labeling offer a hope to find biological markers that allow the study simultaneously the participation of multiple inflammatory proteins in FM patients, allowing the identification of biomarkers. Thus, the use of isobaric tagging will allow shortly to expand the knowledge of pathophysiology in fibromyalgia, helping to identify biomarkers to improve the diagnosis of the FM disease and increase the quality of life of patients and their families.