WO2013170011A2 - Method for determining biospecimen quality - Google Patents

Abstract

A method of assigning an index which indicates the degradation of a type-specific sample, comprising: providing a first specimen of a type-specific sample, subjecting the first specimen to condition(s) which degrade protein(s) in the first specimen; performing surface- enhanced laser desorption/ionization time-of-flight mass spectroscopy analysis on the first specimen to determine protein peak intensities for individual proteins in the first specimen; based on the protein peak intensities, identifying protein(s) in the first specimen as having degraded or not degraded; and assigning an index which is derived from a ratio of the degraded protein(s) to the non-degraded protein(s).

Description

METHOD FOR DETERMINING BIOSPECIMEN QUALITY

[001] This application claims priority to United States Provisional Application Number 61/644,584 filed on May 9, 2012, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[002] It is widely accepted that variable biorepository specimen handling conditions can significantly alter outcomes of clinical research studies, suggesting the need for a metric for sample analyte protein integrity. In line with the NIH and the OBBR Best Practices, it is vital that the integrity of specimens used for biomarker studies are of the highest standard to ensure validity of the data they generate and confidence in the application of new findings to clinical management.

[003] The development of diagnostic tools and the identification of therapeutic targets depend on the availability of biospecimens and their appropriate collection. As known for many years, the collection procedures of biospecimens can have a significant impact on analytical data [1]. Biobanking is a young discipline that is constantly evolving in response to the development of new techniques and new scientific goals. To accomplish the current and future needs of the scientific community, biobanks need to face some essential challenges including an appropriate design, harmonized, rigorous, robust procedures and sustainability; all of these issues need to be achieved in the framework of ethical, legal and social considerations [2]. A preserved human biospecimen allows a researcher an opportunity to study the molecular characteristics of disease and correlate clinical progression with the biology of an individual's disease. With the biorepository so prominently featured in the genomic age, there is a crucial need to undertake research to assure that biospecimen quality is maintained at the highest level possible. A compromised specimen may lead to erroneous results that might result in false reporting, ambiguity and hindrance of the field of study.

SUMMARY OF THE INVENTION

[004] Described herein is a method to discover proteins in biorepository samples to assess the integrity of stored specimens for protein-based biomarker studies, similar to the universally accepted quality metric for RNA, the RNA Integrity Number (RIN). Because certain preanalytical conditions may result in proteolysis and other proteome-associated changes in biorepository samples, this method mimics variation in preanalytical conditions. Further, the method employs surface-enhanced laser desorption time-of-flight mass spectrometry (SELDI-TOF MS) to assess changes in multiple proteins and peptides in a high- throughput manner. Candidate peaks from SELDI spectra of representative sample types were selected and quantified, and these peaks demonstrated differing but reproducible sensitivity to sub-optimal processing and storage The inventors then assigned an index known as a "Sample- specific Protein Integrity Number" (SPIN) to each sample type. The present inventive method can be used to obtain a SPIN index for any type of biological sample. The SPIN index can be used by a biobank or laboratory to assess the quality of biobanked specimens.

[005] In one embodiment, the method of assigning an index which indicates degradation of a type-specific sample, includes: (a) providing a first specimen of a type-specific sample, which first specimen has proteins, (b) subjecting the first specimen to one or more conditions which degrade one or more of the proteins in the first specimen, (c) performing surface- enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF MS) analysis on the first specimen to determine protein peak intensities for individual proteins in the first specimen, (d) based on the protein peak intensities, identifying one or more proteins in the first specimen as having degraded, (e) based on the protein peak intensities, identifying one or more proteins in the first specimen as not having degraded, and (f) assigning an index which is derived from a ratio of the one or more degraded proteins to the one or more non- degraded proteins.

[006] Further, these methods may include providing a second specimen of the type- specific sample, which second specimen has proteins, and assaying the second specimen to determine levels of the one or more degraded proteins and the one or more non-degraded proteins, wherein the levels of the one or more degraded proteins and the one or more non- degraded proteins in the second specimen indicate the amount of degradation of the second specimen.

[007] In some embodiments of the present invention, the protein peak intensities of the one or more degraded proteins may decrease by about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%; and/or the protein peak intensities of the one or more non-degraded proteins may decrease by about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20%.

[008] In another embodiment, the present method also may include (a) identifying the one or more of the degraded proteins and/or non-degraded proteins by tandem mass spectroscopy, ELISA, western blot, or multiplex bead array; and (b) assigning an index which is derived from a ratio for each of three degraded proteins to one non-degraded protein, and averaging the three ratios.

[009] Further, with the present method, the one or more conditions which cause degradation of the one or more proteins may be the passage of time or a change in temperature; the passage of time may be 30, 60, 90, or 120 minutes; the change in temperature may be one or more cycles of freezing and thawing the first specimen; the type- specific sample may be a tissue or a liquid; the type-specific sample may be serum, urine, thyroid tissue, and spinal tissue; and, two, three, four, five, six, seven, eight, nine, or ten proteins may be identified as having degraded.

BRIEF DESCRIPTION OF THE DRAWINGS

[010] Figure 1 depicts the process flow for SPIN candidate protein selection.

[011] Figures 2A-2F show representative spectra, gel view, and cluster plots for dynamic and stable protein peak species as determined by univariate analysis of peak clusters. Figures 2A-2C show dynamic protein M/Z=7785.23. Figures 2D-2F show stable protein

M/Z=3899.35. Serum was incubated at room temperature for 30, 60, or 120 minutes before SELDI analysis.

[012] Figures 3A-3F show a comparison of samples with a "Strong" and "Weak" SPIN. Strong SPIN means specimens processed under ideal conditions, while Weak SPIN means specimens subjected to conditions which result in protein degradation, i.e., the passage of time. A multiplex bead array assay was used to detect IL-2 and IL-6 in urine (Figs. 3B and 3D), IL-2 in thyroid papillary cancer (Fig. 3A) and IL-2 in spinal vertebral tissue (Fig. 3E). In an independent measurement, ELISA was employed to detect insulin c-peptide in serum (Fig. 3C). These results demonstrate analyte-dependent variation with sample type. Fig. 3F shows analyte-dependent variation i.e., percent Weak SPIN analyte concentration versus Strong SPIN analyte concentration, within one sample type (urine).

DETAILED DESCRIPTION OF THE INVENTION

[013] Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.

[014] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also

encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[015] All references, patents, patent publications, articles, and databases, referred to in this application are incorporated herein by reference in their entirety, as if each were specifically and individually incorporated herein by reference. Such patents, patent publications, articles, and databases are incorporated for the purpose of describing and disclosing the subject components of the invention that are described in those patents, patent publications, articles, and databases, which components might be used in connection with the presently described invention. The information provided below is not admitted to be prior art to the present invention, but is provided solely to assist the understanding of the reader.

[016] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, embodiments, and advantages of the invention will be apparent from the description and drawings, and from the claims. The preferred embodiments of the present invention may be understood more readily by reference to the following detailed description of the specific embodiments and the Examples included hereafter.

[017] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry described below are those well-known and commonly employed in the art. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

[018] In this specification and the appended claims, the singular forms "a," "an" and

"the" include plural reference unless the context clearly dictates otherwise.

[019] As used herein, "biological specimen", "biospecimen" and or "specimen" mean any fluid or other material which has been derived from any part of the body of a normal or diseased subject. Biological specimens include, but are not limited to, cells or tissue (e.g., brain tissue, spinal tissue, liver tissue), gametes, blood, serum, plasma, buffy cost, lymph, urine, saliva, tears, sweat, feces, cerebrospinal fluid, milk, amniotic fluid, bile, ascites fluid, pus, and the like, which have been derived from a normal or diseased subject. Biological specimens may be benign, malignant, diseased, or normal. Also included within the meaning of the terms "biological specimen", "biospecimen" and "specimen" is an organ or tissue extract or a culture fluid in which any cells or tissue preparation from a subject has been stored or incubated. Formats for storing biological specimens are known in the art, for example: fresh; frozen, formalin- fixed or paraffin-embedded tissues; histological slides; and floating in a fixative. Methods of obtaining biological samples are well known in the art and may occur concurrently with surgery, autopsy, or routine procedures on a subject.

[020] As used herein, the term "conditions which degrade" mean preanalytical variations that exist in handling a specimen. Preanalytical variations include, for example, variations in sample processing protocols and/or storage conditions, and the presence of and amount of freeze-thaw cycles.

[021] As used herein, the term "degrade", "degraded", and "degradation" refer to the breakdown of a protein by the cleavage of one or more of its peptide bonds, or by other biological, chemical, or environmental action, resulting in simpler substances, such as peptides or amino acids.

[022] As used herein, the terms "protein" is used to refer to a polymer of amino acid residues. The term "amino acid" includes naturally occurring amino acids that are encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, alpha-carboxyglutamate, and O-phosphoserine.

[023] As used herein, the term "protein peak intensity" or "protein peak intensities" means the absolute or relative ion signal intensity or intensities of the protein(s) of interest in a mass spectrum. A classic mass spectrum appears with units of m/z on the x axis and the ion "signal intensity" on the y axis. Absolute ion signal intensity means that the absolute measured intensity of an ion signal is proportional to the amount of sample in the ion source. To derive a quantitative measure, the ion signal intensity measured for the unknown sample is proportionally connected to the ion signal intensity measured for some standard of known amount. That proportionality is apparent in a calibration curve graph, or in the intercept in a method of standard addition. In contrast, relative ion signal intensity means that the most intense (most abundant) peak in the mass spectrum is assigned a value of 100, and all of the other peaks are plotted in the mass spectrum (or tabulated) relative to that value. The actual measured intensity of that base peak has a value, and the other peaks in the mass spectrum are scaled proportionately.

[024] As used herein, the terms "subject" refers to a mammal, preferably a human, who is either normal or is in need of diagnosis and/or treatment for a condition, disorder or disease. The term "patient" refers to a mammal, preferably a human, who is in need of such diagnosis and/or treatment.

[025] As used herein, the term "surface-enhanced laser desorption/ionization time-of- flight mass spectroscopy" (SELDI-TOF-MS) means a bioanalytical technique in mass spectroscopy that is used for the examination of proteins or protein mixtures. SELDI-TOF- MS separates proteins on different solid-phase chromatographic surfaces (ProteinChip) and the proteins are subsequently ionized and detected by TOF-MS. This technique differentiates proteins based on their biochemical or biophysical characteristics.

[026] As used herein, the term "time-of-flight mass spectrometer" (TOF-MS) means a spectrometer that determines an ion's mass-to-charge ratio (m/z) based on how long it takes for that ion to travel down the instrument' s "flight tube" and reach the detector.

[027] As used herein, the term "type- specific sample" and "sample-specific" mean a specific type of sample. Examples of specific types of samples include, but are not limited to: breast, uterus and ovary tissues; colon, stomach, and esophagus tissues; kidney, bladder and prostate tissues; liver, pancreas, and spleen tissues; lung, pharynx, and oral cavity tissues; brain, spinal cord, and peripheral nerves tissues; muscle, skin and soft tissue tissues; thyroid tissue; serum; plasma; lymph; urine; saliva; tears; cerebrospinal fluid; milk; amniotic fluid; bile; ascites fluid; and pus.

[028] Biobanks play an important role in the quest for personalized medicine and improvements in healthcare. However, the biobank must be organized and run properly with procedures and checks in place to ensure that the specimens it contains are of the highest quality. The inventors herein describe a novel methodology to assess the integrity of proteins in biorepository sample types, both fluid and tissue. The present inventive methods may be expanded to virtually every sample type encountered— each having its own protein complement with unique patterns indicative of sample integrity.

[029] Specimen quality, as well as the quality of the analysis obtained from a specimen, depends on the biological and preanalytical variations that exist in a specimen. Biological variation includes sex, age, hormonal status, biological rhythms, exercise, xenobiotics, nutrition, smoking, and genetic factors. Biological variation is beyond the control of the biorepository. However, the biorepository does have control over preanalytical variation. The impact of preanalytical variation is diverse. For specimens of blood, the time delay and the storage temperature prior to processing can influence the extent of clot formation, cross- linking and fibrinolysis. Centrifugation time, force and temperature influence the efficiency of cell separation. For protein expression analysis, knowledge of the preanalytical variation in specimens, across sample-type, is crucial. For example, the activation status of blood cells may change upon exposure to extracellular factors. Environmental solution modifications and contact with synthetic surfaces, during and after phlebotomy, may modify protein expression levels and the extent of posttranslational modifications. The position of the patient during sampling may induce an increase or decrease in the concentration of certain analytes.

[030] Storage-associated preanalytical variation for all types of samples includes the time between sampling and freezing, the storage temperature and protocol, the duration of storage, and the number of freeze-thaw cycles. Previous biospecimen research studies have shown that micromolecular markers such as amino acids, free fatty acids, sodium, cholesterol, triglycerides and vitamin E do not vary significantly after 30 freeze-thaw cycles [3]. Hormones such as estradiol, prolactin, and free and total testosterone are stable in frozen serum or plasma for 3 years at -80°C [4]. Vitamins such as retinol, β-carotene and a- tocopherol are slightly sensitive to freeze-thaw cycles [5]. DNA in whole blood degrades more rapidly at 4°C than at ambient temperature, probably due to increased granulocyte lysis at 4°C [6]. However, greater sensitivity to preanalytical conditions is likely to be experienced by low- abundance proteins, which are likely the biomarkers of tomorrow.

[031] Biobanks, which cryopreserve biological specimens, need to control preanalytical variation and to perform quality control (QC) procedures to determine the extent of preanalytical variation in the specimens and sample-types. Methods for assessing sample and specimen stability are important to QC parameters and procedures. Currently, one part of some biobank quality assurance QC programs is measuring changes in various serum markers according to storage conditions, such as repeated freeze-thaw cycles.

[032] There have been studies that have evaluated how pre-analytical variables for plasma and serum collection affect protein integrity (as determined by liquid

chromatography-mass spectrometry or "LC-MS") with a goal to establish a standard panel of markers to grade blood protein integrity [10]. Due to the cost and availability of a LC-MS, however, this methodology is not available to the average tissue banking facility. Also nano LC-MS and tandem mass spectroscopy (MS/MS) analysis of brain tissues subject to different postmortem time intervals showed that the degradation of a number of proteins started immediately, and that the protein Stathmin 2-20 could serve as a marker of brain sample integrity for this tissue type [11]. However, a more broadly applicable, cost-effective way to screen the quality of the numerous sample types is needed.

[033] SELDI-TOF MS using ProteinChip arrays allows for easy screening of proteins in both tissue and fluid sample types. ProteinChip arrays provide various surface chemistries that enable the capture and analysis of specific subclasses of proteins from tissue and other sample types. SELDI requires very small amounts of a specimen and facilitates rapid screening particularly of low molecular weight (<20 kDa) proteins and peptides.

[034] SELDI analysis has been applied to evaluate archival cytology material for distinct, reproducible protein fingerprints and to demonstrate the potential for specific pathologic diagnoses [7]. This study correlated protein profile fingerprints with renal cancer phenotypes from fine needle aspirates but did not examine variation in protein integrity throughout processing and storage.

[035] Stability of SELDI proteome profiles of urine and serum related to storage conditions also has been evaluated [8]. This study evaluated whether refrigerated storage of serum or urine prior to deep-freezing significantly affected variability across SELDI spectra as a whole; but did not analyze specific mass/charge (m/z) peaks. Further, methods for tissue preservation and storage have been proposed and success of the methods has been confirmed by SELDI analysis [9].

[036] Additionally, SELDI-TOF technology was utilized in a study of reproducibility of results and variability introduced by serum handling in the pre-analytical setting [12]. That study found prolonged transport and incubation at room temperature generated low mass peaks, resulting in distinctions among the protocols. The most and least stringent methods gave the lowest overall peak variances, indicating that proteolysis in the latter may have been nearly complete. For specimens transported on ice, clotting time, storage method, and transit time had little effect. Certain proteins (TR, ApoCI, and transferrin) were unaffected by handling, but others (ITIH4 and hemoglobin β) displayed significant variability. The main objective of that study was to select an optimal protocol for serum collection and handling for a large case-control study which would be feasible for clinical collection.

[037] The present methods use SELDI-TOF-MS technology to develop a sample-specific protein integrity number (SPIN) index that describes the state and thus utility of the specimen for a specific type of sample, irrespective of the processing procedure. ProteinChip SELDI- TOF-MS offers many advantages which make it innovative and highly successful in this setting. It is high-throughput, cost efficient, works with non-digested specimens and is particularly sensitive in analyzing low molecular weight proteins and peptides.

[038] By using SELDI technology, dynamic protein peaks can be selected and the intensities of those peaks used for downstream analytical determinations. Having chosen candidate protein peaks by SELDI technology, type-specific samples can be fractionated by HPLC and peaks in the appropriate fractions confirmed by re-analysis using SELDI. And the candidate protein can be positively identified, quantified, and verified by standard laboratory techniques such as tandem mass spectroscopy (MS/MS), ELISA, western blot, or multiplex bead array. Thus, either absolute or relative ratios can be used for a SPIN index.

[039] SPIN is an index that informs researchers about the quality of the specimens they have in their biobanks; and the present methods can be employed to develop SPIN index for each type of sample. These methods can be used to identify biomarkers that are sensitive to variations in processing protocols, storage conditions, and to the presence of freeze-thaw cycles. Such biomarkers can be used as QC indicators in a biobank quality assurance/QC program.

[040] The SPIN indices can be applied throughout the biorepository and research community to standardize quality measurements for subsequent proteomic analyses and for application of biomarker studies through to translational research, with better personalized patient care as the end goal. SPIN can be used for the global harmonization of collection and pre-analytical procedures.

[041] Generally, the methods of the present invention are directed to developing a SPIN index for any type of sample. Sample types might be in an aqueous form or in the form of a tissue. By way of example, and not as a limitation, tissue sample types could include tissue from the breast, uterus, ovary, colon, stomach, esophagus, kidney, bladder, prostate, liver, pancreas, spleen, lung, pharynx, oral cavity, brain, spinal cord, peripheral nerves, muscle, skin soft tissue, and thyroid; and aqueous sample types could include serum, plasma, lymph, urine, saliva, tears, cerebrospinal fluid, milk, amniotic fluid, bile, ascites fluid, and pus.

[042] Developing a SPIN index for a type-specific sample involves identification of protein(s) which indicate degradation of the type-specific sample, including: obtaining a first specimen of a particular sample-type, and that first specimen includes one or more proteins. The first specimen is subjected to one or more conditions which degrade the protein(s) in the first specimen. In some embodiments of the invention, the condition(s) which cause degradation of the protein(s) are, for example, the passage of time or a change in temperature. In one example, the passage of time may be 30, 60, 90, or 120 minutes. Also, the change in temperature may be one or more cycles of freezing and thawing the first specimen. Due to the conditions of degradation, some of the proteins in the first specimen will degrade, but others will not.

[043] Developing a SPIN index for the type-specific sample also may include using SELDI-TOF MS analysis on the first specimen to identify those "dynamic" protein(s) which have degraded, or have changed due to the conditions of degradation. Two, three, four, five, six, seven, eight, nine, or ten proteins may be identified as having degraded. For example, three dynamic proteins are identified. [044] In one embodiment, surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF MS) analysis is performed on the first specimen to determine protein peak intensities for individual proteins in the first specimen and thereby identify the extent of degradation, if any, in the protein(s) in the first specimen. And based on protein peak intensities produced by the SELDI-TOF MS analysis, one or more proteins in the first specimen is identified as having degraded.

[045] In one embodiment, determining whether a peak intensity is dynamic is based on one or more of the following criteria: (1) the peak is present in all conditions studied, (2) there is decreased peak intensity progressing from control to more rigorous handling conditions, (3) the peak is easily identified and clearly separated from adjacent peaks, (4) the peak intensity is dintinguishable, and/or (5) the p value is <0.05 upon univariate statistical analysis of clustered peaks within one experimental condition. And in some embodiments of the present invention, the protein peak intensities of the degraded protein(s) decreased by about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as caused by the degradation condition(s) to which the first specimen was subjected.

[046] Further, developing a SPIN index for a type-specific sample also includes using SELDI-TOF MS analysis on the first specimen to identify one or more "stable" proteins in the first specimen, i.e., a protein(s) that has not significantly degraded based on analysis of their protein peak intensities. Such stable protein(s) can be used as a control. In one embodiment, a stable peak has a p value that is greater than 0.95. And in some embodiments, the peak intensities of the one or more non-degraded (stable) proteins decreased by about 0%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20%.

[047] The SPIN index for a specific type of sample can be derived from the protein peak intensities of the dynamic and the stable proteins. In one embodiment, a SPIN index is assigned to each dynamic protein. That SPIN index is calculated by dividing the peak intensity of each dynamic protein peak (observed at the end time-point) by the peak intensity of the same stable protein. Also, SPIN indexes each of the three dynamic protein peaks can be averaged for each experimental condition and expressed as mean SPIN for a sample-type.

[048] Further, the degraded (dynamic) and non-degraded (stable) proteins can be positively identified to determine the name and/or type of the protein. And the degraded and non-degraded proteins can be verified. Methods for such identification and verification include, for example, tandem mass spectroscopy, ELISA, western blot, or multiplex bead array.

[049] Moreover, the degraded and non-degraded proteins selected by the present methods can form the basis for a simple assay to measure the SPIN (specimen integrity) of a specimen of a specific sample type. For example, a sample type-specific assay can be developed for use by a biobank, irrespective of a specimen's pre-analytical conditions. This assay may include providing a second specimen of the type-specific sample, which second specimen has proteins, and assaying the second specimen to determine levels of the degraded protein(s) and non-degraded protein(s), wherein the levels of the degraded protein(s) and the non-degraded protein(s) in the second specimen indicate the amount of degradation of the second specimen. For example, such a type-specific assay could be based on lateral flow "dip stick" technology [15], ELISA, or a multiplex bead assay to quantitate (e.g., determine expression levels) for the proteins included in the SPIN index for a specific type of sample.

[050] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLES

[051] The following examples illustrate various embodiments of the invention and are not intended to limit the scope of the invention.

[052] Example 1: Materials and Methods for Examples 2-4

[053] Serum, Urine and Tissue Samples. Serum, urine, thyroid papillary cancer tissue, and periprosthetic spinal vertebral tissue were collected according to institutional HIC policies and processed according to the Beaumont Research Institute's BioBank Standard Operating Procedures (SOPs) with various modifications designed to introduce pre-analytical variability which may be encountered in biorepository laboratories (Figure 1). For serum, blood was drawn into red-top vacutainer tubes and allowed to clot for 30 minutes at room temperature in a vertical position. Within one hour of collection, the blood was centrifuged at room temperature at 1800 x g followed by serum removal and aliquoting into freezing tubes. Freezing was mediated by the use of a thermo conductive freezing system (Biocision LLC, Mill Valley, CA) and subsequently stored at -80°C. Urine was collected into a specimen cup and sampled into a vacutainer tube without preservatives, centrifuged to remove particulate matter, aliquoted into freezing tubes, frozen and then stored at -80°C. Thyroid tissue samples were snap-frozen within thirty minutes of surgical removal and stored at -80°C. Periprosthetic spinal vertebral tissue was placed immediately into RNA later (Ambion Biosystems, Austin, TX) and then minced to homogeneity with a razor blade, divided into equal aliquots and frozen at -80°C.

[054] Introduction of pre-analytical variability. Frozen aliquots of serum or urine were thawed and subjected to pre-analytical variations that may be encountered commonly in a biorepository. These included varying freeze-thaw cycles or extended incubation at room temperature. Extracts were made from spinal disc tissue by homogenizing in 5 volumes (w:v) of phosphate buffered saline (PBS) containing 0.5% Tween 20. Homogenates were cleared by centrifugation at 17K x g for 10 minutes in a microfuge at 4°C. Supernatants were transferred to fresh microcentrifuge tubes and subjected to a second centrifugation. The protein concentration of the supernatant from this second centrifugation was determined by the BCA protein assay (ThermoFisher Scientific, Rockford, IL). Protein concentrations of the extracts were then normalized to 2.5ug/ul by the addition of homogenization buffer. Aliquots of the normalized extracts were then exposed to varying freeze-thaw cycles or extended incubations at room temperature. Thyroid tissue was minced and mixed to homogeneity while still frozen and divided into equal aliquots which were the subjected to varying conditions. After treatment, tissue was solubilized in U9 buffer (Biorad, Hercules, CA) consisting of 9M urea, 2% CHAPS, 50mM Tris-HCl, 1 % DTT, pH 9.0 with the aid of a micropestle. After centrifuging, the protein concentration in the cleared extract was determined and normalized with the addition of U9 buffer.

[055] SELDI analysis. Discovery proteomics was performed using a ProteinChip

SELDI-TOF MS, Enterprise Edition (Biorad Laboratories, Hercules, CA). The ProteinChip used for this study was the CM10 (weak cation exchange). Briefly, specimens were thawed on ice then centrifuged to clear, after which 20 μΐ of supernatant was denatured by the addition of 30μ1 of U9 buffer. The specimens were incubated on ice for 30 minutes. Next,

2μ1 of specimen was spotted in duplicate, after randomization, onto an array with 4μ1 of pre- spotted low stringency binding buffer (0.1 M sodium acetate, PH 4.0). Proteins were allowed to bind in a humid chamber for 60 minutes after which liquid was removed and the spots allowed to dry. Next, Ι μΐ of saturated sinapinic acid (SPA) in 50% acetonitrile and 0.5% trifluoroacetic acid was applied as an energy absorbing matrix and allowed to crystallize by solvent evaporation for 10 minutes at room temperature. After another addition/drying of Ι μΐ

SPA, the arrays were ready for SELDI data acquisition. The array spots were bombarded under two laser intensity conditions (low energy 1600nJ and high energy 3500nJ) to more closely analyze lower and higher mass ranges. Each spot was divided into 50 partitions (4 pixels per partition), and each selected pixel was bombarded 12 times (the first two being

"warming shots at the selected energy + 10%). For the 1600nJ condition, energy source was set at 25kV, positive ions, mass range 0-20,000 Da, focus mass = 5,000 Da, matrix attenuation = 1000, sampling rate = 800MHz. Under 3500nJ bombardment, settings were adjusted to mass range = 0-200,000 Da, focus mass 16,000, and matrix attenuation = 5,000. This resulted in the generation of numerous individual spectra. Multiple aliquots of a pooled reference specimen consisting of a mixture of all the specimens were also spotted onto the ProteinChips for quality control, standardization and normalization purposes.

[056] Spectra analysis. Analysis was performed using ProteinChip Data Manager 3.5 (Biorad Laboratories, Inc. Hercules, CA, USA). Once accumulated, spectra were subject to peak detection, and qualified mass peaks were then defined by first creating and applying mass calibration equations using Biorad' s All-in-One peptide standards (low energy condition) or All-in-One protein standards (high energy condition). Peak intensity calculations were subsequently adjusted after baseline subtraction (fitting width set at 10 times the expected peak width using the smoothing function set at 25 points for the low mass range and 10 points for the high mass range). Next, noise was reduced by adjusting the average filter to 0.2 times the expected peak width. Peak intensities within a condition were subsequently normalized to total ion current to compensate for spectrum-to-spectrum variations, and outlying spectra were removed from the analysis. Spectra were then aligned to a reference-pool specimen with a normalization factor of 1. Peak clustering was then carried out to group peaks of similar mass across multiple spectra using automatic first-pass peak detection settings of signal to noise ratio of > 2.0. Peak clusters were further defined using second-pass peak detection (signal-to-noise ratio >2, valley depth = 2.0) with minimum peak threshold set at 15% and a mass window approximately 0.1% of the peak mass.

Estimated peaks were added to ensure that every spectrum is represented in the cluster. After peak clustering, peaks were subjected to statistical analysis with p- values calculated across each group. Following univariate analysis and P value calculation, differences in spectra patterns and peak intensities were studied. Significant peak clusters (P value < 0.05) were then considered candidates for future identification and protein integrity focus. Interim SPIN numbers (ratio of non-stable to stable SELDI peak intensities) were assigned.

[057] ELISA and multiplex bead array quantitation. To demonstrate the effect of sub- optimal specimen handling (as indicated by the SPIN index) on the outcomes of laboratory analytical determinations, quantitative analysis in the form of analyte- specific ELISAs or multiplex cytokine bead arrays were then performed according to the manufacturer' s protocols. For each sample-type (thyroid papillary cancer, urine, serum, and spinal vertebral tissue), specimens were divided, with a first part subject to ideal handling conditions and a second part subjected to conditions that degrade proteins, i.e., the passage of one-half hour of time. SELDI analysis showed a strong SPIN for the first part and a weak SPIN for the second part (Figs. 3A-3E). Analytes for the samples were selected for their known sensitivity to specimen handling. Cytokine (IL-Ιβ, IL-2, IL-6, and IL-8) concentrations were determined by multiplex bead array (R&D Systems, Minneapolis, MN). Insulin C-peptide was determined by ELISA (Millipore, Billerica, MA). Analyte-specific ELISAs or multiplex cytokine bead arrays were used to quantify analyte levels in the first and second parts.

[058] Example 2: Selection of Peaks from SELDI Spectra

[059] To determine the effect of varying collection and processing conditions SELDI analysis was performed on the manipulated specimens and spectra across each sample type were compared. Under the conditions studied, 363 peak clusters were revealed in serum, 273 in urine, 137 in thyroid tissue extract, and 123 in spinal tissue extract. For each sample type in the study, four peaks (m/z) representing protein species were selected from SELDI proteomic profiles. Three of these were selected because they displayed dynamic changes in peak intensities. Specimens were exposed to extended room temperature incubations or multiple free-thaw cycles. The peak representing a fourth protein was chosen as a stable internal reference (i.e. the peak intensity remained the same in all conditions). Selection criteria for the dynamic protein peaks include: (1) peak is present in all conditions studied, (2) decreased peak intensity progressing from control to more rigorous handling conditions, (3) peak was easily identified and clearly separated from adjacent peaks, (4) intensity greater than 30, and (5) p value <0.05 upon univariate statistical analysis of clustered peaks within one experimental condition. The selected stable control peak demonstrated a p value >0.95 (Figure 2).

[060] Example 3: SPIN Assignment in Each Sample Type

[061] After selecting a stable protein peak and three dynamic protein peaks that demonstrated decreasing intensity trends using SELDI analysis and statistics software, a SPIN index for each dynamic protein was calculated. This was done by dividing the reported peak intensity of each dynamic protein peak (observed at the end time-point) by the peak intensity of the stable protein. SPIN indexes from the three dynamic protein peaks were averaged for each experimental condition and expressed as mean SPIN (Table 1). Table 1 shows SPIN indices of representative sample/conditions. SPIN indices for each dynamic protein were calculated by dividing the peak intensity of each dynamic protein by the peak intensity of a stable reference protein. [062] Table 1 :

sample condition dynamic #1 dynamic #2 dynamic #3 Mean + SD

M/Z=7785.23 M/Z=8709.72 M/Z=9309.73

serum 1 120' rt 0.77 1.10 0.77 0.88 + 0.19 scrum 2 Ι Ι n ( 1. 6 0. ⁽ 1.74 Ο 4 + 0.02 serum 3 120' rt i. & 0.82 0.78 + 0.04 scrum 4 1 0^' n 1 1.53 ⁽ I.S3 ^{( )}.« ⁾ 0.65 + 0. 1 6 serum 5 120' rt 0.42 0.97 0.46 0.62 + 0.31

M/Z=2941.24 M/Z=4969.19 M/Z=11664.84

thyroid 1 120' rt 0.20 1.73 1.12 1.02 + 0.77

thyroid 3 120' rt 0.42 0.03 0.02 0.16 + 0.23

M/Z=1480.74 M/Z=2535.11 M/Z=6267.82

urine 1 o/n rt 0.73 0.20 1.00 0.64 + 0.41 urine 2 o, n n 0.55 0.27 0.25 0.36 + 0. 1 7 urine 3 o/n rt 0.16 1.05 0.02 0.41 + 0.56 uri ne 4 o/n ri 0.67 0.20 0.25 0.67 + 0.26 urine 5 o/n rt 0.15 0.52 0.37 0.35 + 0.19

M/Z=2293.30 M/Z=4934.78 M/Z=12595.44

spine 1 120' rt 0.31 0.63 0.42 0.45 + 0.16 spi ne 2 1 0^' n 0.22 l .oo 1 .25 O.S2 + 0.54 spine 3 120' n 0.80 0.75 0.67 0.74 + 0.07

[063] Example 4: Correlation between SPIN and analytical results

[064] Select assays performed on samples subjected to handling variation revealed a correlation between SPIN and absolute analyte quantitation. These results suggest that SPIN can be utilized to predict sample quality and resulting proteomic analysis performance.

Cytokines (e.g., IL-Ι β, IL-2, IL-6, and IL-8) are known to have a short half-life in-vivo and are also subject to rapid degradation in-vitro following specimen collection if appropriate storage and handling procedures are not adapted. In several cytokine studies, these same factors involving specimen collection, processing and storage have been shown to be critical for achieving accurate and reproducible results. Most of these published reports on cytokine stability and storage are related to serum or plasma sample types [13]. Cytokine levels in all four sample types decreased with decreasing SPIN index (Figures 3A-3E). Conversely, slight increases were observed in serum insulin c-peptide levels within the time course of the study and thus had and inverse relationship to the SPIN index suggesting that increased incubation times under the conditions studied could lead to increasing pro-peptide processing and result in artificially inflated values for this analyte. Similar increases were observed previously in other studies [14]. These analytical artifacts were introduced as a result of specimen handling during processing and storage and demonstrate the importance of having a SPIN reference index. Additionally, different analytes within one sample type were more dependent on handling and storage conditions and displayed varied relationships to SPIN (Figure 3F).

REFERENCES

1. Bernard AM: Biokinetics and stability aspects of biomarkers: recommendations for application in population studies. Toxicology 1995;101 :65-71.

2. Riegman PH, Morente MM, Betsou F, et al.: Biobanking for better healthcare. Mol Oncol 2008;2:213-222.

3. Paltiel L, Ronningen KS, Meltzer HM, et al.: Evaluation of Freeze Thaw Cycles on stored plasma in the Biobank of the Norwegian Mother and Child Cohort Study. Cell Preserv Technol 2008;6:223-230.

4. Bolelli G, Muti P, Micheli A, et al. : Validity for epidemiological studies of long-term cryoconservation of steroid and protein hormones in serum and plasma. Cancer Epidemiol Biomarkers Prev 1995;4:509-513.

5. Wahrendorf J, Hanck AB, Munoz N, et al.: Vitamin measurements in pooled blood samples. Am J Epidemiol 1986;123:544-550.

6. Cuypers HT, Bresters D, Winkel IN, et al.: Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus. J Clin Microbiol 1992;30:3220-3224.

7. Fetsch PA, Simone NL, Bryant-Greenwood PK, et al.: Proteomic evaluation of archival cytologic material using SELDI affinity mass spectrometry: potential for diagnostic applications. Am J Clin Pathol 2002;118:870-876.

8. Traum AZ, Wells MP, Aivado M, et al. : SELDI-TOF MS of quadruplicate urine and serum samples to evaluate changes related to storage conditions. Proteomics 2006;6: 1676- 1680.

9. Bouamrani A, Ternier J, Ratel D, et al.: Direct-tissue SELDI-TOF mass spectrometry analysis: a new application for clinical proteomics. Clin Chem 2006;52:2103-2106. 10. Becker CH, Bern M: Recent developments in quantitative proteomics. Mutat Res;722: 171-182.

11. Skold K, Svensson M, Norrman M, et al. : The significance of biochemical and molecular sample integrity in brain proteomics and peptidomics: stathmin 2-20 and peptides as sample quality indicators. Proteomics 2007;7:4445-4456.

12. Timms JF, Arslan-Low E, Gentry-Maharaj A, et al.: Preanalytic influence of sample handling on SELDI-TOF serum protein profiles. Clin Chem 2007;53:645-656.

13. Panicker G, Meadows KS, Lee DR, et al.: Effect of storage temperatures on the stability of cytokines in cervical mucous. Cytokine 2007;37:176-179.

14. Evans MJ, Livesey JH, Ellis MJ, Yandle TG: Effect of anticoagulants and storage temperatures on stability of plasma and serum hormones. Clin Biochem 2001;34:107-112.

15. Haeberle S, Zengerle R: Microfluidic platforms for lab-on-a-chip applications. Lab Chip 2007;7:1094-1110.

Claims

WHAT IS CLAIMED IS:

1. A method of assigning an index which indicates degradation of a type-specific sample, comprising:

providing a first specimen of a type-specific sample, which first specimen has proteins,

subjecting the first specimen to one or more conditions which degrade one or more of the proteins in the first specimen,

performing surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF MS) analysis on the first specimen to determine protein peak intensities for individual proteins in the first specimen,

based on the protein peak intensities, identifying one or more proteins in the first specimen as having degraded,

based on the protein peak intensities, identifying one or more proteins in the first specimen as not having degraded, and

assigning an index which is derived from a ratio of the one or more degraded proteins to the one or more non-degraded proteins.

2. The method of claim 1 , wherein the index is derived from a ratio for each of three degraded proteins to one non-degraded protein and averaging the three ratios.

3. The method of claim 1, wherein the protein peak intensities of the one or more degraded proteins decreased by about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%.

4. The method of claim 1 , wherein the protein peak intensities of the one or more non-degraded proteins decreased by about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20%.

5. The method of claim 1, further comprising identifying the one or more of the degraded proteins by tandem mass spectroscopy, ELISA, western blot, or multiplex bead array.

6. The method of claim 5, wherein the identification of the one or more degraded proteins is by tandem mass spectroscopy.

7. The method of claim 1, further comprising identifying the one or more of the non-degraded proteins by tandem mass spectroscopy, ELISA, western blot, or multiplex bead array.

8. The method of claim 7, wherein the identification of the one or more non- degraded proteins is by tandem mass spectroscopy.

9. The method of claim 1, further comprising;

providing a second specimen of the type- specific sample, which second specimen has proteins, and

assaying the second specimen to determine levels of the one or more degraded proteins and the one or more non-degraded proteins,

wherein the levels of the one or more degraded proteins and the one or more non- degraded proteins in the second specimen indicate the amount of degradation of the second specimen.

10. The method of claim 1, wherein the one or more conditions which cause degradation of the one or more proteins is either the passage of time or a change in temperature.

11. The method of claim 10, wherein the one or more conditions which cause degradation of the one or more proteins is a change in temperature.

12. The method of claim 10, wherein the passage of time is 30, 60, 90, or 120 minutes.

13. The method of claim 12, wherein the change in temperature is one or more cycles of freezing and thawing the first specimen.

14. The method of claim 1, wherein the type-specific sample is a tissue or a liquid.

15. The method of claim 14, wherein the type-specific sample is a tissue.

16. The method of claim 14, wherein the type-specific sample is selected from the group consisting of serum, urine, thyroid tissue, and spinal tissue.

17. The method of claim 1, wherein two, three, four, five, six, seven, eight, nine, or ten proteins are identified as having degraded.

18. The method of claim 17, wherein three proteins are identified as having degraded.