Design considerations for proteomic
reference materials
David M. Bunk
Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, MD, USA
Received: April 13, 2010 Revised: July 22, 2010 Accepted: August 25, 2010 In order to improve the repeatability, comparability, and accuracy of MS-based proteomic
measurements, there has been considerable international effort to develop appropriate reference materials. Although the majority of reference materials are developed to support measurement quality of routine assays, the development of reference materials for a diverse and changing research field such as proteomics represents unique challenges. In order to define common measurement components and common features of typical proteomic samples, the metrology underpinning proteomics must be considered due to the diversity and changing nature of the field. Reference materials can then be designed around common aspects in order to produce reference materials with the broadest applicability. Reference materials are needed to support both qualitative and quantitative proteomic measurements, involving different design consid-erations. Consensus and validated statistical approaches to describe the confidence in qualitative measurement, such as protein identification, needs to be established. Common sources of measurement bias also need to be considered in proteomic reference material design.
Keywords:
Accuracy / Animal proteomics / Comparability / Reference materials
1
Introduction
Proteomic research has quickly become a significant contributor to furthering basic understanding in the biolo-gical sciences as well as contributing to more applied fields such as biofuels, drug discovery, and medical diagnoses. Like many research areas before it, the early days of proteomics suffered from significant measurement quality issues. Studies published by the HUPO and the Association
of Biomolecular Research Facilities (ABRF) demonstrated significantly inter- and intra-laboratory comparability problems with both qualitative and quantitative proteomic measurement [1–4] using MS. As these early measurement problems had the potential to erode the confidence in proteomics research, there has been considerable interna-tional effort to improve measurement quality. Starting back in 2002, both the HUPO and the ABRF have established initiatives aimed to improve the comparability and repeat-ability of proteomic measurement protocols, informatics, and to develop reagents and standards for the proteomics community. There have been efforts to improve the quality of published proteomic research through the establishment of publication standards [5]. In 2006, the US National Cancer Institute (NCI) launched the Clinical Proteomics Technology Assessment for Cancer (CPTAC) program which aims to improve MS-based proteomics measurement quality to facilitate biomarker discovery and proteomic research in cancer (http://proteomics.cancer.gov/programs/ CPTAC/). In addition to these efforts, proteomic reagent and reference material development by HUPO Abbreviations: ABRF, Association of Biomolecular Research
Facilities; CPTAC,Clinical Proteomics Technology Assessment for Cancer;ID-MS,isotope dilution MS;MRM,multiple reaction monitoring; NCI, National Cancer Institute; NIST, National Institute of Standards and Technology; SRM,Standard Refer-ence Material
Correspondence:Dr. David Bunk, Analytical Chemistry Division, National Institute of Standards and Technology, NIST 100 Bureau Drive, MS 8392 Gaithersburg, MD 20899-8392, USA E-mail:[email protected]
[3], ABRF (http://www.sigmaaldrich.com/life-science/ proteomics/mass-spectrometry/ups1-and-ups2-proteomic. html), and CPTAC (http://antibodies.cancer.gov/ reagents/) has been an important step towards improving measurement quality.
Reference materials are widely used in measurement quality assurance for routine assays in areas such as food and nutritional testing, environmental analysis, and clinical diag-nostics. Although the primary use of reference materials is to support measurement accuracy, reference materials also support measurement comparability as control materials for intra- and inter-laboratory performance assessment. In addi-tion, reference materials can be used in the development and validation of new assays. The design of reference materials to support a routine assay is usually straightforward, dictated by the nature of samples being assayed and the measurement approaches used. For example, the National Institute of Stan-dards and Technology (NIST) Standard Reference Material (SRM) 967a (Creatinine in Frozen Human Serum) is a pooled human serum material with certified concentrations of crea-tinine which is used to support clinical serum creacrea-tinine assays. The pooled human serum matrix and the levels of creatinine concentrations present in SRM 967a were chosen to best represent the characteristics of patient samples commonly measured by routine creatinine assay. In addition, the analy-tical methods used by NIST to measure the creatinine concentration in SRM 967 match the selectivity and specificity of most routine clinical diagnostic methods [6]. In contrast to an application such as routine diagnostic creatinine assays, designing reference materials to support measurement quality in proteomics is not straightforward.
Proteomics is an evolving research field with a highly varied field of application. Proteomic research changes depending on the research goals, the laboratory performing the measurements, and over time as techniques and instrumentation improve. This evolution makes designing reference materials for MS-based proteomics a significant challenge as the intended use of a proteomic reference material is difficult to clearly define. Nevertheless, providing well-characterized, high-quality reference materials to the proteomics community could provide important tools to further the improvements in measurement quality.
2
Reference material design and
production
Before discussing the specific issue of proteomic reference materials, there are fundamental issues regarding reference material design and production that should first be discus-sed. As was mentioned above, reference materials can be used to support measurement accuracy, as ‘‘trueness’’ controls in proficiency testing, or as tools for the develop-ment and/or validation of new measuredevelop-ment approaches. The starting point for reference material development is always defining the intended use(s).
Although the intended use of a reference material is to support a specific analytical measurement, reference mate-rials can either support part of a measurement process or the entire process. A measurement process can usually be broken down into a series of sequential sub-processes, such as sample processing, analytical measurement, and data analysis (Fig. 1). It is, therefore, possible to design a refer-ence material whose intended use is to support the entire measurement process, often referred to as a vertical refer-ence material. Vertical referrefer-ence materials are surrogates of routine samples and should closely resemble them in terms of matrix and analyte concentration; how close a vertical reference material needs to be to a true sample depends on several factors, including the specificity of the routine assay and the sensitivity of the routine assay to changes in sample matrix and the presence of interferences. NIST SRM 967a, discussed above, is an example of a vertical reference material. On the contrary, horizontal reference materials support one or more components within a measurement process such as the analytical measurement. As such, a horizontal reference material will most closely resemble the sample in the form present during the measurement sub-process. For a horizontal reference material designed for a specific analytical measurement, the reference material would have the same matrix complexity and analyte concentration as a sample that had undergone the same degree of sample processing used in the routine assay. An example of a horizontal reference material is NIST SRM 2389a, an aqueous mixture of amino acids which is designed to support accuracy in the quantification of amino acids. As a mixture of amino acids in a simple matrix, NIST SRM 2389a resembles samples of purified proteins or peptides after the hydrolysis step in quantitative amino acid
Sample Vertical Reference Material Reference Material Horizontal Reference Material Horizontal Reference Material Horizontal Sample Processing Analytical Measurement Data Analysis Measurement Result
Figure 1. Relationship of vertical and horizontal reference materials to an analytical measurement process
analysis. As such, SRM 2389a (amino acids in 0.1 mol/L hydrochloric acid) supports only the analytical measurement of amino acids but not the ‘‘up-stream’’ sample processing. A characteristic of a horizontal reference material is that it may have broad applicability when a measurement sub-process is used in a several different assays. NIST SRM 2389a has broad applicability to most methods for amino acid measurement such as ion or gas chromatography of derivatized or underivatized amino acids.
Regardless of the application to which it is directed, a reference material, first and foremost, must be both stable and homogeneous. It would provide little benefit to support measurement accuracy if the composition (either that of the analyte(s) or matrix) of a reference material changed from vial to vial or if the reference material displayed instability (of either the analyte(s) or matrix) over time. Therefore, the homogeneity and stability of a reference material must be rigorously evaluated. Homogeneity refers to both the homogeneity within a single vial of a reference material and the homogeneity between vials. Intra-vial inhomogeneity is not often observed with reference materials that are in liquid or frozen liquid form but can be a problem with solid reference materials such as in soils for environmental test-ing or foodstuff for nutritional analysis. For a carefully prepared reference material, the inter-vial inhomogeneity is typically more significant than intra-vial inhomogeneity. When reference materials are created in a large batch and then aliquotted into hundreds or thousands of vials, inter-vial homogeneity must be considered. As it takes time to aliquot a reference material into vials, during which time the material may not be maintained at a temperature in which it is the most stable ( 801C, for example), it is possible that short-term instability can impact analytes within vials filled early compared with vials filled at the end. Additionally, when an analyte is a ‘‘sticky’’ molecule, such as a protein, if the aliquotting apparatus has not been sufficiently passi-vated with the sample, there is the possibility that adsorptive losses may impact vials filled earlier more than vials filled later. Because of these and other concerns, inter-vial homogeneity must always be validated through measure-ment and never be assumed.
The stability of a reference material must also be eval-uated, including short-term and long-term stabilities. The short-term stability evaluation aims to determine if changes in the composition of the reference material are possible during shipment from the reference material producer to the end-user. Assessment of long-term stability assures that no drift occurs in the value(s) assigned to reference mate-rials during the lot lifetime which allows the reference material to be used to determine measurement compar-ability over time.
Another important aspect of reference material produc-tion is the value assignment of analytes of interest. In the value assignment of reference materials, the aim is to provide the true value to the concentration or identification of the analyte of interest by maximizing accuracy and
minimizing measurement uncertainty. When a primary reference measurement procedure [7] (http:// www.cstl.nist.gov/nist839/NIST_special_publications.htm (accessed July 2010)) (also called a definitive method) is used to assign value to a reference material, the highest degree of analytical accuracy is achieved, resulting in a ‘‘certified’’ reference material (at NIST, ‘‘Standard Refer-ence Material’’ is the term used for ‘‘certified referRefer-ence material’’). A primary reference measurement procedure is defined by the International Bureau of Weights and Measures [8] as ‘‘a method having the highest metrological properties, whose operation can be completely described and understood, for which a complete uncertainty state-ment can be written down in terms of Syste`me Interna-tional units.’’ A primary reference measurement procedure aims to achieve the highest accuracy possible through a thorough evaluation of measurement bias and uncertainty to achieve the ‘‘true’’ value of the analyte(s) of interest. When a primary reference measurement proce-dure is not available for value assignment or when the analytical rigor of a primary reference measurement procedure is not necessary for the intended use, a refer-ence material is produced for which analyte concentration is not certified. The confidence in the accuracy of non-certified analyte concentration is lower than for non-certified analytes and formulating a complete uncertainty state-ment for non-certified values is generally not possible.
3
Reference materials for qualitative
proteomics
A fundamental purpose of proteomic measurements is protein identification. Measurement to identify proteins of interest often leads to further investigations in which these proteins are quantified. An error in protein identification can completely undermine the success of the quantitative measurement. Therefore, measurement quality assurance in qualitative proteomics is necessary to provide a solid foundation for future proteomic research. Reference mate-rials could be an important tool to improve measurement quality assurance in qualitative proteomics. Designing reference materials for this task does provide several unique challenges.
As the application of proteomics for protein identification is so diverse, designing reference materials to suite all applications is not feasible. However, it is possible to consider some vertical reference materials that may have use for several applications and horizontal reference materials that could be cross-cutting for a large number of applica-tions. NIST is currently working on two reference materials to support qualitative proteomics, a vertical reference material and a horizontal reference material.
The NCI CPTAC program, in which NIST has been involved since its inception, has demonstrated the utility of using the Saccharomyces cerevisiaeyeast proteome as
perfor-mance standard for evaluating LC-MS/MS perforperfor-mance in data-dependent acquisition mode [9]. Additionally, the yeast proteome has been used as a model proteome in many fundamental proteomic investigations. The yeast proteome is attractive as a reference material for several reasons including its availability, proteomic complexity, and range of protein concentrations. With regard to clinical proteomic research aiming to discover biomarkers in human tissue, the proteo-mic complexity and protein concentration range of most human tissue is similar to that of the yeast proteome. A yeast proteome reference material may be a successful surrogate to a wider range of proteomes when the proteomic complexity of the proteome of interest is similar to that of yeast or fractio-nation of the proteome of interest reduces the protein complexity to a level similar to yeast.
Building on the study done by the CPTAC investigators, NIST developed a yeast proteome reference material as a vertical reference material, a material that can be carried through an entire proteomics workflow from sample preparation through data analysis. The yeast protein study material prepared by NIST for CPTAC investigation had undergone sample preparation, including trypsin digestion, before use in CPTAC inter-laboratory studies [9]. The goals of the CPTAC studies focused more on the analytical measurement and data analysis but not on sample preparation. Therefore, the sample preparation of the yeast used for CPTAC studies was performed at NIST to remove any potential lab-to-lab variability in sample preparation. For use as a vertical reference material, the yeast proteome reference material differs from the material used in CPTAC investigations in that minimal sample processing will be performed such that the reference material will consist of a frozen aqueous solution of intact yeast proteins. Users of the NIST yeast reference material will be able to choose the sample preparation that best matches their current practice. The moderate complexity of the yeast proteome could allow this reference material to be effectively used as a quality control material for the protein separation techniques employed in proteomic sample preparation, such as elec-trophoresis and chromatography. A less complex protein mixture might not challenge the capacity of typical separa-tion methods, whereas a very complex proteome could overwhelm separation capacity.
As the protein identifications resulting from the CPTAC investigations using S. cerevisiae yeast were obtained using a common sample preparation procedure and a common instrument type operated using a prescribed standard operating procedure, the identified protein values can only be considered method-dependent values. Without a thorough investigation of potential bias introduced by the common sample preparation, analytical measurement, and data analysis protocol, the proteins identified through any single measurement method cannot strictly be considered ‘‘true’’ values. A different approach is needed for the value assignment of a yeast proteome reference material. No primary reference measurement procedure exists to assign protein identifications to a yeast reference material. In
the absence of a primary reference measurement procedure, a wide variety of proteomic measurement approaches must be employed to assign consensus protein identifications to the reference material. If data obtained using a variety of sample preparation, analytical measurement and data analysis approa-ches are combined to obtain ‘‘consensus’’ protein identification; bias introduced by any one component of the proteomic measurement should have less impact on consensus values. For example, if trypsin digestion is the only method of proteolysis used in sample preparation for the measurement of a yeast reference material, the proteins identified will not include proteins that are resistant to trypsin digestion or contain few arginine or lysine residues. Using a variety of proteolysis method for the yeast sample preparation would increase the likelihood that proteins not amenable to trypsin digest would be identified in a consensus process. Similarly, employing measurements on many instrument platforms would reduce any instrument bias to protein identification. In fact, when no primary measurement procedure exists to certify the concen-tration of analytes in reference materials, a variety of carefully chosen routine methods can be used to accurately determine the analyte’ concentration [10]. A similar consensus approach could be applied to qualitative measurements such as protein identification.
The majority of reference materials for chemical analysis are used to support quality in quantitative measurements. Measurements for protein identification provide a nominal property (i.e.the name of the protein) rather than numerical values. Although the chemical metrology community has established statistical ‘‘best practices’’ for evaluating quan-titative results and reference measurement procedures for quantitative measurement, nothing comparable exists for qualitative measurements. To evaluate the overall uncer-tainty (or confidence) of identified proteins from a consen-sus approach, the statistical framework to weight the confidence of data from each approach and express the confidence of the consensus values would need to be developed. To produce reference materials with certified nominal properties, statistical approaches must also be developed to evaluate homogeneity and stability with regard to nominal properties. Prior to certifying protein identifi-cations in the yeast reference material, the statistical approaches to assess the overall uncertainty of identifica-tions must be clearly defined and vetted within the chemical metrology community. Currently, similar issues are being addressed for the certification of nucleic acid reference materials in which the certified property is the identity of a specific gene or the sequence of a nucleic acid.
Another outcome of the CPTAC program has been to highlight the role that a reference material could play in evaluating instrument performance in qualitative MS/MS. NIST prepared a trypsin digest of a mixture of 20 human proteins (referred to as the ‘‘NCI-20’’ mixture) for CPTAC inter-laboratory studies aimed at evaluating repeatability and comparability of qualitative proteomics [11]. Although it was originally believed that a simple mixture of 20 proteins was not
appropriately complex for evaluating proteomic measurement quality, the data obtained from LC-MS/MS of the NCI-20 digest proved otherwise. Analysis of the NCI-20 digest data showed that many metrics related to the performance of chromatography and data-dependent MS/MS could be identi-fied [12]. The NCI-20 mixture digest proved to be a starting point for the development of a similar reference material at NIST, a horizontal reference material designed to support LC-MS/MS instrument performance evaluation.
The NCI-20 digest mixture resulted in a moderately complex mixture of peptides present at a wide range of relative concentrations. Both the peptide complexity and range of concentrations were beneficial characteristics for the use of the mixture as an instrument performance material. For the design of a reference material for instrument performance evaluation, there are changes from the NCI-20 mixture digest that will improve the utility of the material. Using synthetic peptides in the mixture rather than peptides derived from proteolysis should result in a better-characterized reference material and will allow more control in selecting the range of peptide concentrations in the reference material. Compared with peptides derived from protein digestion, the composition of a mixture of synthetic peptide would be much easier to prepare consistently from batch to batch, an important consideration for reference material production. Using synthetic peptides would also provide better control of the complexity of the mixture. Data-dependent acquisition of a complex peptide mixture is often characterized by undersampling if the complexity of the mixture exceeds the sampling capabilities of the MS instru-mentation. For an effective performance material, the data obtained from MS/MS analysis of the material should reflect only instrument performance and not be adversely affected by limitations to the sampling rate of the instrument. Hence, although a complex mixture of peptides best represents samples being analyzed in proteomic experimentation, a mixture that is too complex is not desirable for the intended use. Taking all of this into consideration, NIST is developing a peptide-based horizontal reference material for use in the evaluation of instrument performance in data-dependent LC-MS/MS. This reference material will be a mixture of synthetic peptides with a mixture complexity and concentration range that is designed to challenge both the LC and the MS sampling space appro-priately. Use of this type of reference material to establish instrument performance in data-dependent-based LC-MS/MS should improve the reliability of proteomic data across a variety of qualitative applications. In addition to LC-MS/MS, a peptide mixture such as this could be applicable to the proteomic measurements using matrix-assisted laser desorption ionization MS.
4
Reference materials for quantitative
proteomics
Quantitative proteomic measurements fall into two cate-gories: relative and absolute quantitation. In relative
quan-titation, the goal of the proteomic investigation is typically the determination of differences in protein abundances between proteome samples in different states, such as the human plasma proteome from healthy and diseased indi-viduals. In the common proteomic experimental design, two proteome samples under examination are each chemically labeled with a different labeling reagent, the differentially labeled samples are blended together, and the analytical MS measurement of the blended sample is performed to measure the difference in signal between labeled compo-nents [13, 14]. The quantity measured in relative quantita-tion is the difference in the concentraquantita-tions of proteins of interest between samples. This contrasts to absolute quan-tification [15] in which the determination of the concentra-tions of proteins of interest in a single proteome sample is determined in absolute units (i.e.g/L, mol/L).
Quantitative bias in both types of measurements makes it a challenge to effectively design and use reference materials to evaluate measurement accuracy in quantitative proteo-mics. Accuracy in relative quantification can be biased due to the limitations of the dynamic range of the mass spec-trometers used. The magnitude of the potential bias will be instrument dependent and also dependent on the magni-tude of the differences in the measured ion intensities. When this measurement bias is present, the values of protein concentration differences of reference materials might not be achievable using routine proteomic methods, undermining the effective use of the reference materials to support measurement accuracy.
In most applications of absolute quantification in MS-based proteomics, proteolytic peptides are quantified as surrogate measurands for the proteins of interest. One of the approaches is to quantify proteolytic peptides using multiple reaction monitoring (MRM) methods of MS in which isotopically labeled synthetic peptides are used as internal standards. This is an extension to peptide analytes of isotope-dilution MS (ID-MS), which has been in use for several decades to accurately quantify small organic compounds [16]. In the proteomic application of ID-MS, if the proteolysis process is not complete, the measured concentration of proteolytic peptides will not be the same as their precursor protein, introducing a source of quantitative bias. The magnitude of the bias will depend on the method of proteolysis and the protein substrate. Therefore, quanti-tative proteomics based on MRM-based measurement of proteolytic peptides yield results which are method depen-dent and not ‘‘true’’ protein concentration values. However, it is important to point out that these measurements are often fit to achieve the intended research goals which do not require absolute accuracy but do require consistency of results. The consequence of this measurement bias to reference material design is that a peptide-based reference material would be more suited to support measurement accuracy in MRM-based measurements than a protein-based reference material. MRM-based ID-MS measurements used routinely now in proteomics have the potential to accurately
measure peptide concentration and, therefore, a peptide-based reference material with accurately assigned peptide concentrations could be effectively used to assess routine proteomic measurements.
In the design of reference materials to support quanti-tative proteomic measurements, NIST is expanding upon the effort to produce reference materials for qualitative measurements. The yeast reference material would be a good platform to develop a reference material for relative quantitation. By growing yeast under two different growth conditions, two yeast proteomes could be produced which displayed differential protein expression for select proteins. In addition to providing values of identified proteins, the concentrations of select proteins which displayed differ-ential expression would be provided for the two yeast proteome reference material. The peptide performance mixture designed for evaluating qualitative instrument performance could be augmented by the addition of isoto-pically labeled analogues of selected peptides and providing certified concentrations of labeled and unlabeled peptides. The peptide mixture reference material could then serve to assess the accuracy of MRM-based proteomic measurement of peptides.
5
Concluding remarks
Efforts to improve measurement quality in MS-based proteo-mics have not only raised awareness of these issues within the scientific community but are leading to meaningful solutions, such as reference materials. As proteomic research is dynamic and is applied with a wide scope, challenges exist to producing relevant reference materials. However, through an assessment of the common measurement challenges of both qualitative and quantitative proteomics and by considering the funda-mental metrology of proteomics, reference materials can be designed. NIST is currently implementing designs for two reference materials to support measurement quality in proteomics. When these reference materials are in place, additional reference material resources can be developed for the proteomics community.
The authors have declared no conflict of interest.
6
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