Interlaboratory Comparison of Antibody-Free LC-MS/MS Measurements of C-peptide and Insulin.

BACKGROUND: The enhanced precision and selectivity of liquid chromatography-tandem mass spectrometry (LC-MS/MS) makes it an attractive alternative to certain clinical immunoassays. Easily transferrable work flows could help facilitate harmonization and ensure high-quality patient care. We aimed to evaluate the interlaboratory comparability of antibody-free multiplexed insulin and C-peptide LC-MS/MS measurements. METHODS: The laboratories that comprise the Targeted Mass Spectrometry Assays for Diabetes and Obesity Research (TaMADOR) consortium verified the performance of a validated peptide-based assay (reproducibility, linearity, and lower limit of the measuring interval [LLMI]). An interlaboratory comparison study was then performed using shared calibrators, de-identified leftover laboratory samples, and reference materials. RESULTS: During verification, the measurements were precise (2.7% to 3.7%CV), linear (4 to 15 ng/mL for C-peptide and 2 to 14 ng/mL for insulin), and sensitive (LLMI of 0.04 to 0.10 ng/mL for C-peptide and 0.03 ng/mL for insulin). Median imprecision across the 3 laboratories was 13.4% (inter-quartile range [IQR] 11.6%) for C-peptide and 22.2% (IQR 20.9%) for insulin using individual measurements, and 10.8% (IQR 8.7%) and 15.3% (IQR 14.9%) for C-peptide and insulin, respectively, when replicate measurements were averaged. Method comparison with the University of Missouri reference method for C-peptide demonstrated a robust linear correlation with a slope of 1.044 and r2 = 0.99. CONCLUSIONS: Our results suggest that combined LC-MS/MS measurements of C-peptide and insulin are robust and adaptable and that standardization with a reference measurement procedure could allow accurate and precise measurements across sites, which could be important to diabetes research and help patient care in the future.

New Views of Old Proteins: Clarifying the Enigmatic Proteome.

All human diseases involve proteins, yet our current tools to characterize and quantify them are limited. To better elucidate proteins across space, time, and molecular composition, we provide a >10 years of projection for technologies to meet the challenges that protein biology presents. With a broad perspective, we discuss grand opportunities to transition the science of proteomics into a more propulsive enterprise. Extrapolating recent trends, we describe a next generation of approaches to define, quantify, and visualize the multiple dimensions of the proteome, thereby transforming our understanding and interactions with human disease in the coming decade.

How many human proteoforms are there?

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

The human proteome project: Current state and future direction.

After successful completion of the Human Genome Project (HGP), HUPO has recently officially launched a global Human Proteome Project (HPP) which is designed to map the entire human protein set. Given the presence of about 30% undisclosed proteins out of 20,300 protein gene products, a systematic global effort is necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP groups employ the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge base. The HPP participants will take advantage of the output and cross-analyses from the ongoing HUPO initiatives and a chromosome-based protein mapping strategy, termed C-HPP with many national teams currently engaged. In addition, numerous biologically-driven projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents and tools for protein studies and analyses, and a stronger basis for personalized medicine. HUPO urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.

The human proteome project: current state and future direction.

After the successful completion of the Human Genome Project, the Human Proteome Organization has recently officially launched a global Human Proteome Project (HPP), which is designed to map the entire human protein set. Given the lack of protein-level evidence for about 30% of the estimated 20,300 protein-coding genes, a systematic global effort will be necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP research groups will use the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge bases. The HPP participants will take advantage of the output and cross-analyses from the ongoing Human Proteome Organization initiatives and a chromosome-centric protein mapping strategy, termed C-HPP, with which many national teams are currently engaged. In addition, numerous biologically driven and disease-oriented projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents, and tools for protein studies and analyses, and a stronger basis for personalized medicine. The Human Proteome Organization urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.

Plasma protein biomarkers predict the development of persistent autoantibodies and type 1 diabetes 6 months prior to the onset of autoimmunity.

Type 1 diabetes (T1D) results from autoimmune destruction of ß cells. Insufficient availability of biomarkers represents a significant gap in understanding the disease cause and progression. We conduct blinded, two-phase case-control plasma proteomics on the TEDDY study to identify biomarkers predictive of T1D development. Untargeted proteomics of 2,252 samples from 184 individuals identify 376 regulated proteins, showing alteration of complement, inflammatory signaling, and metabolic proteins even prior to autoimmunity onset. Extracellular matrix and antigen presentation proteins are differentially regulated in individuals who progress to T1D vs. those that remain in autoimmunity. Targeted proteomics measurements of 167 proteins in 6,426 samples from 990 individuals validate 83 biomarkers. A machine learning analysis predicts if individuals would remain in autoimmunity or develop T1D 6 months before autoantibody appearance, with areas under receiver operating characteristic curves of 0.871 and 0.918, respectively. Our study identifies and validates biomarkers, highlighting pathways affected during T1D development.

A HUPO test sample study reveals common problems in mass spectrometry-based proteomics.

We performed a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in liquid chromatography-mass spectrometry-based proteomics. We distributed an equimolar test sample, comprising 20 highly purified recombinant human proteins, to 27 laboratories. Each protein contained one or more unique tryptic peptides of 1,250 Da to test for ion selection and sampling in the mass spectrometer. Of the 27 labs, members of only 7 labs initially reported all 20 proteins correctly, and members of only 1 lab reported all tryptic peptides of 1,250 Da. Centralized analysis of the raw data, however, revealed that all 20 proteins and most of the 1,250 Da peptides had been detected in all 27 labs. Our centralized analysis determined missed identifications (false negatives), environmental contamination, database matching and curation of protein identifications as sources of problems. Improved search engines and databases are needed for mass spectrometry-based proteomics.

The Evolving Landscape of Autoantigen Discovery and Characterization in Type 1 Diabetes.

Type 1 diabetes (T1D) is an autoimmune disease that is caused, in part, by T cell-mediated destruction of insulin-producing ß-cells. High risk for disease, in those with genetic susceptibility, is predicted by the presence of two or more autoantibodies against insulin, the 65-kDa form of glutamic acid decarboxylase (GAD65), insulinoma-associated protein 2 (IA-2), and zinc transporter 8 (ZnT8). Despite this knowledge, we still do not know what leads to the breakdown of tolerance to these autoantigens, and we have an incomplete understanding of T1D etiology and pathophysiology. Several new autoantibodies have recently been discovered using innovative technologies, but neither their potential utility in monitoring disease development and treatment nor their role in the pathophysiology and etiology of T1D has been explored. Moreover, neoantigen generation (through posttranslational modification, the formation of hybrid peptides containing two distinct regions of an antigen or antigens, alternative open reading frame usage, and translation of RNA splicing variants) has been reported, and autoreactive T cells that target these neoantigens have been identified. Collectively, these new studies provide a conceptual framework to understand the breakdown of self-tolerance, if such modifications occur in a tissue- or disease-specific context. A recent workshop sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases brought together investigators who are using new methods and technologies to identify autoantigens and characterize immune responses toward these proteins. Researchers with diverse expertise shared ideas and identified resources to accelerate antigen discovery and the detection of autoimmune responses in T1D. The application of this knowledge will direct strategies for the identification of improved biomarkers for disease progression and treatment response monitoring and, ultimately, will form the foundation for novel antigen-specific therapeutics. This Perspective highlights the key issues that were addressed at the workshop and identifies areas for future investigation.

A multiprotein nuclear complex connects Fanconi anemia and Bloom syndrome.

Bloom syndrome (BS) is a genetic disorder associated with dwarfism, immunodeficiency, reduced fertility, and an elevated risk of cancer. To investigate the mechanism of this disease, we isolated from human HeLa extracts three complexes containing the helicase defective in BS, BLM. Interestingly, one of the complexes, termed BRAFT, also contains five of the Fanconi anemia (FA) complementation group proteins (FA proteins). FA resembles BS in genomic instability and cancer predisposition, but most of its gene products have no known biochemical activity, and the molecular pathogenesis of the disease is poorly understood. BRAFT displays a DNA-unwinding activity, which requires the presence of BLM because complexes isolated from BLM-deficient cells lack such an activity. The complex also contains topoisomerase IIIalpha and replication protein A, proteins that are known to interact with BLM and could facilitate unwinding of DNA. We show that BLM complexes isolated from an FA cell line have a lower molecular mass. Our study provides the first biochemical characterization of a multiprotein FA complex and suggests a connection between the BLM and FA pathways of genomic maintenance. The findings that FA proteins are part of a DNA-unwinding complex imply that FA proteins may participate in DNA repair.

Novel SWI/SNF chromatin-remodeling complexes contain a mixed-lineage leukemia chromosomal translocation partner.

The SWI/SNF family of chromatin-remodeling complexes has been discovered in many species and has been shown to regulate gene expression by assisting transcriptional machinery to gain access to their sites in chromatin. Several complexes of this family have been reported for humans. In this study, two additional complexes are described that belong to the same SWI/SNF family. These new complexes contain as many as eight subunits identical to those found in other SWI/SNF complexes, and they possess a similar ATP-dependent nucleosome disruption activity. But unlike known SWI/SNFs, the new complexes are low in abundance and contain an extra subunit conserved between human and yeast SWI/SNF complexes. This subunit, ENL, is a homolog of the yeast SWI/SNF subunit, ANC1/TFG3. Moreover, ENL is a fusion partner for the gene product of MLL that is a common target for chromosomal translocations in human acute leukemia. The resultant MLL-ENL fusion protein associates and cooperates with SWI/SNF complexes to activate transcription of the promoter of HoxA7, a downstream target essential for oncogenic activity of MLL-ENL. Our data suggest that human SWI/SNF complexes show considerable heterogeneity, and one or more may be involved in the etiology of leukemia by cooperating with MLL fusion proteins.

Acrylamide--a cysteine alkylating reagent for quantitative proteomics.

Mass spectrometry-based relative quantification of proteins is often achieved by the labeling of two samples with isotopically light and heavy reagents. The intensities of the ions with different masses, but same chemical properties, can be reliably used for determining relative quantities. Several strategies of labeling with various weakness and strength and degrees of complexity have been described. In this chapter, we describe a simple and inexpensive protein-labeling procedure based on the use of acrylamide and deuterated acrylamide as a cysteine alkylating reagent. Gel electrophoresis is one of the most commonly used techniques for analyzing/visualizing proteins, thus, we emphasize the use of acrylamide as a labeling procedure for quantifying proteins isolated by one- and two-dimensional polyacrylamide gel electrophoresis.

Quantitative proteomics using mass spectrometry.

The use of stable isotopes as internal standards in mass spectrometry has opened a new era for quantitative proteomics. Depending on the point at which the label is introduced, most procedures can be classified as in vivo labeling, in vitro pre-digestion labeling or in vitro post-digestion labeling. In vivo labeling has been used for cells that can be grown in culture and has the advantage of being more accurate. The pre-digestion and post-digestion labeling procedures are suitable for all types of sample including human body fluids and biopsies. Several new mass spectrometric strategies mark significant achievements in determining relative protein concentrations and in quantifying post-translational modifications. However, further technology developments are needed for understanding the complexity of a dynamic system like the proteome.

Quantitative proteomics using mass spectrometry.

The use of stable isotopes as internal standards in mass spectrometry has opened a new era for quantitative proteomics. Depending on the point at which the label is introduced, most procedures can be classified as in vivo labeling, in vitro predigestion labeling or in vitro post-digestion labeling. In vivo labeling has been used for cells that can be grown in culture and has the advantage of being more accurate. The pre-digestion and post-digestion labeling procedures are suitable for all types of sample including human body fluids and biopsies. Several new mass spectrometric strategies mark significant achievements in determining relative protein concentrations and in quantifying post-translational modifications. However, further technology developments are needed for understanding the complexity of a dynamic system like the proteome.

The mitochondrial Italian Human Proteome Project initiative (mt-HPP).

Mitochondria carry maternally inherited genetic material, called the mitochondrial genome (mtDNA), which can be defined as the 25th human chromosome. The chromosome-centric Human Proteome Project (c-HPP) has initially focused its activities addressing the characterization and quantification of the nuclear encoded proteins. Following the last International HUPO Congress in Boston (September 2012) it was clear that however small the mitochondrial chromosome is, it plays an important role in many biological and physiopathological functions. Mutations in the mtDNA have been shown to be associated with dozens of unexplained disorders and the information contained in the mtDNA should be of major relevance to the understanding of many human diseases. Within this paper we describe the Italian initiative of the Human Proteome Project dedicated to mitochondria as part of both programs: chromosome-centric (c-HPP) and Biology/Disease (B/D-HPP). The mt-HPP has finally shifted the attention of the HUPO community outside the nuclear chromosomes with the general purpose to highlight the mitochondrial processes influencing the human health. Following this vision and considering the large interest and evidence collected on the non-Mendelian heredity of Homo sapiens associated with mt-chromosome and with the microbial commensal ecosystem constituting our organism we may speculate that this program will represent an initial step toward other HPP initiatives focusing on human phenotypic heredity.

The human proteome - a scientific opportunity for transforming diagnostics, therapeutics, and healthcare.

A National Institutes of Health (NIH) workshop was convened in Bethesda, MD on September 26-27, 2011, with representative scientific leaders in the field of proteomics and its applications to clinical settings. The main purpose of this workshop was to articulate ways in which the biomedical research community can capitalize on recent technology advances and synergize with ongoing efforts to advance the field of human proteomics. This executive summary and the following full report describe the main discussions and outcomes of the workshop.

A method to identify and simultaneously determine the relative quantities of proteins isolated by gel electrophoresis.

Gel electrophoresis is often used for the primary analysis and purification of proteins, and peptide mapping by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a widely used technique for the rapid identification of unknown proteins. The identification is usually obtained by digesting the protein with an enzyme and matching the masses of the proteolytic peptides with those of each protein in a sequence database. Another important aspect in many proteomic experiments is the determination of the relative protein quantities (e.g. comparison between control and altered states). Usually, this is obtained by comparing the spot intensities of two independent gels. This procedure is time-consuming and not very accurate. Recently, several methodologies using isotope labeling of proteins for quantitative proteomic studies have been introduced (e.g. using ICAT reagents or growing cells in isotopically enriched nutrients). However, none of these methodologies is foolproof and there is still the need for simple and inexpensive alternatives for determining the relative quantities of proteins. Previously, we showed that a mixture of acrylamide and deuterated acrylamide could be used as cysteine alkylating reagent prior to electrophoresis, improving the coverage and the confidence of the protein identification procedure (Sechi S, Chait BT. Anal. Chem. 1998; 70: 5150). Here we show that a similar approach can be used to obtain relative quantitation at the femtomole level of proteins isolated by gel electrophoresis. Deuterated acrylamide is used to alkylate the cysteines in one sample and regular acrylamide is used to alkylate the cysteines in the second sample. The two samples are then mixed together in a 1:1 ratio and the relative protein quantities are determined from the ion intensity ratios of the two cysteine-containing peptides isotopic envelopes (regular/deuterated). The analysis of several proteins mixed in different ratios is reported showing that this approach can reliably be used for protein identification and quantification. Briefly, a simple and inexpensive method for quantifying and simultaneously identifying proteins isolated by gel electrophoresis using MALDI-MS is presented.

The ATRX syndrome protein forms a chromatin-remodeling complex with Daxx and localizes in promyelocytic leukemia nuclear bodies.

ATRX syndrome is characterized by X-linked mental retardation associated with alpha-thalassemia. The gene mutated in this disease, ATRX, encodes a plant homeodomain-like finger and a SWI2/SNF2-like ATPase motif, both of which are often found in chromatin-remodeling enzymes, but ATRX has not been characterized biochemically. By immunoprecipitation from HeLa extract, we found that ATRX is in a complex with transcription cofactor Daxx. The following evidence supports that ATRX and Daxx are components of an ATP-dependent chromatin-remodeling complex: (i) Daxx and ATRX can be coimmunoisolated by antibodies specific for each protein; (ii) a proportion of Daxx cofractionates with ATRX as a complex of 1 MDa by gel-filtration analysis; (iii) in extract from cells of a patient with ATRX syndrome, the level of the Daxx-ATRX complex is correspondingly reduced; (iv) a proportion of ATRX and Daxx colocalize in promyelocytic leukemia nuclear bodies, with which Daxx had previously been located; and (v) the ATRX complex displays ATP-dependent activities that resemble those of other chromatin-remodeling complexes, including triple-helix DNA displacement and alteration of mononucleosome disruption patterns. But unlike the previously described SWI/SNF or NURD complexes, the ATRX complex does not randomize DNA phasing of the mononucleosomes, suggesting that it may remodel chromatin differently. Taken together, the results suggest that ATRX functions in conjunction with Daxx in a novel chromatin-remodeling complex. The defects in ATRX syndrome may result from inappropriate expression of genes controlled by this complex.