CN111435130A

CN111435130A - SRM and DIA assays for clinical cancer assessment

Info

Publication number: CN111435130A
Application number: CN202010024974.9A
Authority: CN
Inventors: 托德·A·哈姆布拉夫; 法比奥拉·切基; K·M·斯科特
Original assignee: Nantomics LLC
Current assignee: Nantomics LLC
Priority date: 2019-01-11
Filing date: 2020-01-10
Publication date: 2020-07-21
Also published as: KR20200087701A; CA3067536A1; JP2020128976A

Abstract

The present invention provides methods for quantifying the amount of CB L, FPGS, HSP90A, HSP90B, INSR, MC L1 and/or TrkA protein in a formalin-fixed tissue/cell biological sample quantification may be performed by quantifying the amount of one or more fragment peptides derived from the protein in a protein digest using mass spectrometry.

Description

SRM and DIA assays for clinical cancer assessment

Cross reference to related patent applications

This patent application claims the benefit of U.S. provisional patent application No. 62/791,495 filed on 2019, month 1, day 11, the entire contents of which are incorporated herein by reference.

Technical Field

Background

Protein expression levels of one or more proteins selected from the group consisting of CB L, FPGS, HSP90A, HSP90B, INSR, MC L1 and TrkA in patient tumor tissue are determined by quantifying the specific peptide derived from a subsequence of each full-length protein each peptide is detected using mass spectrometry-based Selective Reaction Monitoring (SRM) (also referred to as Multiple Reaction Monitoring (MRM) (herein referred to as SRM assay)) or by Data Independent Acquisition (DIA).

The candidate peptide for developing a single SRM/MRM assay for a single protein could theoretically be any single peptide resulting from a complete protease digestion, e.g., trypsin digestion, of the entire full-length protein. However, surprisingly, many peptides are not suitable for reliable detection and quantification of any given protein-indeed, for some proteins, no suitable peptide has been found. Thus, it is not possible to predict which peptide is most favorable by the SRM/MRM assay for a given protein, and therefore a specifically defined assay characteristic for each peptide must be empirically found and determined. This is particularly true when identifying the optimal SRM/MRM peptide for analysis in protein lysates, such as liquid lysates from formalin-fixed paraffin-embedded tissues. The SRM/MRM assay described herein assigns one or more protease-digested peptides (trypsin-digested peptides) to each protein, thereby finding each peptide to be a favorable peptide for the SRM/MRM assay, particularly a peptide prepared from formalin-fixed patient tissue. The peptides may also be used in Data Independent Acquisition (DIA) assays to detect expression in relative quantification.

Quantitation is relative or absolute. When absolute quantitation is required, the measured level of each peptide is compared to a known amount of a labeled reference peptide having the same amino acid sequence as the measured peptide. The peptides are unique to a particular protein, and thus one peptide molecule is derived from one protein molecule, and thus the quantitative level of the peptide allows quantification of the entire protein from which the peptide is derived. Measurement of protein expression can be used for diagnosis of cancer, staging of cancer, prognosis of cancer progression, predicting the likelihood of clinical response to various cancer treatments and therapies, and the like.

Disclosure of Invention

SRM/MRM and DIA assays are provided that can be used to detect and quantify the levels of specific proteins in proteome lysates prepared directly from tumor tissue of cancer patients. These proteins can be used to inform the selection of cancer therapy and as part of a treatment regimen. The SRM/MRM and DIA assays described herein can be used to develop personalized molecular profiles of tumor cells directly in the tumor tissue of a patient. Once the expression state of these proteins is determined, specific therapeutic agents can be administered to the patient, whereby these agents can interact with the proteins detected and measured by the assays described herein and inhibit or enhance the function of these proteins to kill tumor cells. In addition, therapeutic agents that induce the cancer patient's own immune system to kill the patient's own tumor cells can be administered to the cancer patient as part of this treatment regimen. Both therapeutic agents specifically targeting tumor-associated proteins and immune system-directed therapeutic agents can be directly matched to the molecular profile of cancer patients, as determined by the present invention, providing personalized strategies for targeted and immune-based cancer therapy.

Methods are provided for detecting and/or quantifying specific fragment peptides from one or more proteins selected from CB L, FPGS, HSP90A, HSP90B, INSR, MC L, and trka directly in patient tissue, wherein a therapeutically effective amount of a cancer therapeutic is administered to a patient or subject from which a biological sample is obtained, wherein the amount of the cancer therapeutic and/or the amount of the cancer therapeutic administered is based on the detection and/or amount of any one or more (multiple) fragment peptides of the one or more proteins, wherein the cancer therapeutic is an immunomodulatory cancer therapeutic that functions to initiate, enhance, manipulate, and/or otherwise modulate the patient's immune response to attack and kill tumor cells in the patient.

In particular, methods for detecting and measuring human biological samples are provided (examples)Such as formalin-fixed tissue samples), wherein the method comprises detecting and quantifying by mass spectrometry the amount of one or more protein fragment peptides in a protein digest prepared from a biological sample, wherein the amount is a relative or absolute amount, and calculating the level of one or more proteins in the sample

protocol) was prepared.

Mass spectrometry methods can include tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, orbital trap mass spectrometry, mixed ion trap/quadrupole mass spectrometry, MA L DI-TOF mass spectrometry, MA L DI mass spectrometry, and/or time-of-flight mass spectrometry methods for determining the absolute amount of one or more proteins can be, for example, Selective Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), Intelligent Selective Reaction Monitoring (iSRM), Parallel Reaction Monitoring (PRM), and/or multiple selective reaction monitoring (mSRM). Mass spectrometry methods can be data-independent acquisition methods.

The tissue used to prepare the digests can be paraffin-embedded tissue, and can be obtained from a tumor, such as a primary tumor or a secondary tumor.

A method of quantifying one or more proteins comprises comparing the amount of a fragment peptide in a biological sample to the amount of the same fragment peptide in a different and separate biological sample. A further method for quantifying one or more proteins comprises comparing the amount of fragment peptides in a biological sample to that of fragments from other and different proteins in the same biological sampleAnd comparing the other and different fragment peptides. A further method of quantifying one or more proteins comprises determining the amount of fragment peptide in a biological sample by comparison to a known amount of an added internal standard peptide having the same amino acid sequence. The internal standard peptide may be an isotopically labeled peptide, which may include one or more peptides selected from the group consisting of¹⁸O、¹⁷O、³⁴S、¹⁵N、¹³C、²A heavy stable isotope of H or a combination thereof.

In these methods, detecting and quantifying at least one protein fragment peptide in the protein digest indicates the presence of the corresponding protein in the patient or subject and is associated with cancer. The method may further comprise correlating the results of detecting and quantifying the one or more fragment peptides or the amount of the corresponding protein to a diagnostic histology/stage/grade/status of the cancer. Correlating the results of detecting and quantifying at least one fragment peptide with the diagnostic histology/stage/grade/status of the cancer may be combined with detecting and/or quantifying the amount of other proteins or fragment peptides from other proteins in multiplex format to provide additional information about the diagnostic histology/stage/grade/status of the cancer.

Detailed Description

Methods are provided for measuring the levels of proteins CB L, FPGS, HSP90A, HSP90B, INSR, MC L1 and TrkA in patient tissue (e.g., formalin-fixed tissue). the measured levels of each protein, alone or in combination, can be used as a diagnostic tool as part of an improved method of cancer treatment.

Measured protein

CB L (also known as FRA11B, NS LL and RNF55) is an E3 ubiquitin-protein ligase (E3 ubiquitin-protein ligase) involved in cell signaling and protein ubiquitination CB L functions as an E3 ligase and is therefore capable of catalyzing the formation of covalent bonds between ubiquitin and its protein substrates (typically receptor tyrosine kinases) CB L functions as a negative regulator of many signaling pathways triggered by activation of cell surface receptors and recognizing activated receptor tyrosine kinases including KIT, F L T1, FGFR2, PDGFRA, frbb, EGFR, CSF1R, EPHA8 and KDR, resulting in termination of cell signaling CB L recognizes membrane-bound HCK, SRC and other kinases of the SRC family and mediates ubiquitination and degradation of them CB L is involved in signal transduction and regulates bone resorption and osteoclastogenesis, which are essential for bone resorption and differentiation.

FPGS (also known as poly-gamma-glutamate synthase (plg) and polyglutamate synthase (plg)) are proteins that catalyze the conversion of folate to polyglutamic acid derivatives, which allow for the concentration of folate compounds in cells and the intracellular retention of these cofactors, which are important substrates for most folate-dependent enzymes (folate-dependent enzymes) involved in one-carbon transfer reactions involved in purine, pyrimidine and amino acid synthesis. Unsubstituted reduced folate is a preferred substrate. FPGS also plays a role in the metabolism of Methotrexate (MTX) to polyglutamic acid.

Heat shock protein 90A (HSP90A) and heat shock protein 90B (HSP90B) are nearly identical proteins with identical functions, acting as chaperones to assist other proteins in properly folding, stabilizing proteins against heat stress and aiding protein degradation. These proteins also stabilize many other proteins required for tumor growth, which is why inhibitors of HSP90A and HSP90B have been investigated as anticancer drugs. Heat shock proteins, as a class, are the most highly expressed cellular proteins in all species and generally play a role in protecting cells when stressed by high temperature, accounting for 1-2% of total protein in unstressed cells. However, when the cells are heated, the proportion of heat shock proteins increases to 4-6% of the cellular proteins. HSP90A protein has 2 isoforms, both of which are found in the cytoplasm. Expression of HSP90 AA is inducible, whereas HSP90AB is constitutively expressed. HSP90B protein has only one form and is located in the endoplasmic reticulum. The drug targeting heat shock protein 90A and heat shock protein 90B showed good effect in clinical test. HSP90B has also been identified as one of the autoantigen biomarkers and targets associated with human ovarian autoimmune disease leading to ovarian failure and thus infertility.

The insulin receptor is encoded by A single gene, INSR, which is alternately spliced during transcription (alternative splicing) to produce IR-A or IR-B isoforms, downstream post-translational events of either isoform lead to the formation of proteolytically cleaved α and β subunits, which upon binding are ultimately capable of homo-or heterodimerization (homo-dimerization) to produce an approximately 320kDA disulfide-linked insulin receptor.

MC L1 (also known as myelogenous cell leukemia 1(myeloid cell leukemia 1), a regulator of apoptosis of the BC L2 family, and a regulator of apoptosis of the BC L2 family) are proteins in the Bcl-2 protein family two different isoforms of MC L1 have been identified, a longer gene product produces a longer form of the protein (isoform 1) that enhances cell survival by inhibiting apoptosis, while an alternatively spliced shorter gene product (isoform 2) promotes apoptosis and induces death.

TrkA (also known as tropomyosin receptor kinase a) (Trk receptor kinase a) is a member of the Trk receptor family of tyrosine kinases that regulates synaptic strength and plasticity in the mammalian nervous system Trk receptors influence neuronal survival and differentiation through several signaling cascades, however, activation of these receptors also has a significant effect on the functional properties of neurons, activation of Trk receptors through neurotrophic factor (neurotrophin) binding can lead to activation of signaling cascades, thereby promoting cell survival and other functional regulation, TrkA has the highest affinity for binding of nerve growth factor (nerve growth factor) (NGF), NGF is important in both local and nuclear effects, regulating growth cone, motility and expression of biosynthetic genes encoding neurotransmitter enzymes TrkA dimerizes in response to ligand binding, other tyrosine kinase receptors are also so these dimers phosphorylate each other and enhance the catalytic activity of kinases that influence neuronal growth and differentiation by activating different signaling cascades, three known mitogen kinase (mapik) activation pathways (mapik-kinase) lead to the final mitogen kinase activation of tumor cell activation pathways (PI-kinase) and mitogen kinase activation of cAMP-specific cAMP kinase elements.

Each of these proteins is a target for cancer therapeutics in current clinical practice or clinical development, and thus, determining the expression level of each of these proteins in patient tumor cells can be used as part of the process of making decisions regarding cancer therapeutics.

Measurement method

The following methods provide quantitative proteomics-based assays that can be used to quantify each measured protein in formalin-fixed tissue from cancer patients. For example, data from the assay may be used to make treatment decisions for improved cancer treatment.

As described in more detail below, proteins can be measured by SRM analysis or by the DIA method. SRM analysis can be used to measure the relative or absolute quantitative levels of specific peptides from each measured protein, whereas DIA can only measure the relative levels of the protein. Both assays provide a means to measure the amount of each protein in a given protein preparation obtained from a biological sample by mass spectrometry. More specifically, the assay can measure these peptides directly in complex protein lysate samples prepared from cells obtained from patient tissue samples (e.g., formalin-fixed cancer patient tissue). Methods of preparing protein samples from formalin-fixed tissue are described in U.S. Pat. No. 7,473,532, the contents of which are incorporated herein by reference in their entirety. The method described in U.S. Pat. No. 7,473,532 can be conveniently performed using liquid reagents and protocols obtained from Expression Pathology, inc (Rockville, MD).

Formalin-fixed paraffin-embedded tissue (FFPE) is the most widespread and most advantageous form of tissue from cancer patients, including tumor tissue. Formaldehyde/formalin fixation of surgically excised tissue is by far the most common method of preserving cancer tissue samples worldwide and is a well-established practice in standard pathology practice. Aqueous formaldehyde is known as formalin. "100%" formalin consists of a saturated formaldehyde solution in water (about 40% by volume or 37% by mass) and a small amount of a stabilizer (usually methanol) that limits oxidation and degree of polymerization. The most common way to preserve tissues is to soak whole tissues in aqueous formaldehyde (commonly referred to as 10% neutral buffered formalin) for a period of time (8 to 48 hours), then embed the fixed whole tissues in paraffin and store them at room temperature for a long period of time. Molecular analysis methods for analyzing formalin-fixed cancer tissues are probably the most accepted and most commonly used methods for analyzing tissues of cancer patients.

The results of the SRM or DIA assay can be used to correlate the accurate and precise quantitative levels of each specific protein in a specific tissue sample (e.g., a cancer tissue sample) of a patient or subject from which the tissue (biological sample) is collected and stored. This not only provides diagnostic information about the cancer, but also allows a physician or other medical professional to determine the appropriate treatment for the patient. Such assays that provide diagnostically and therapeutically important information about the level of protein expression in diseased tissues or other patient samples are called companion diagnostic assays (companion diagnostic assays). For example, such assays may be designed to diagnose the stage or extent of cancer and determine the therapeutic agent to which the patient is most likely to respond.

The assays described herein measure the relative or absolute levels of a particular unmodified peptide from a given protein, and may also measure the absolute or relative levels of a particular modified peptide from each of the given proteins. Examples of modifications include phosphorylated and glycosylated amino acid residues present on the peptide.

The relative quantitative level of each protein was determined by the SRM or DIA method. For SRM assays, SRM characteristic peak areas (e.g., characteristic peak areas or integrated fragment ion intensities) of individual fragment peptides derived from proteins in different samples are compared. Alternatively, multiple SRM characteristic peak areas of multiple characteristic peptides, each peptide having its own specific SRM characteristic peak, may be compared to determine the relative protein content in one biological sample as compared to the same protein content in one or more additional or different biological samples. Thus, under the same experimental conditions, the amount of a particular peptide from a protein of interest, and thus the amount of a given protein, in 2 or more biological samples is determined relative to the same peptide. Furthermore, the relative quantification of one or more given peptides from a given protein in a single sample can be determined by comparing the characteristic peak area of a peptide to the characteristic peak area of another and different peptide from one or more different proteins in the same protein preparation from a biological sample by the SRM method. Thus, the amount of a particular peptide from a given protein, as well as the amount of that protein, are determined relative to each other in the same sample. These methods yield quantification of a single peptide or multiple peptides from a given protein to said another peptide or multiple peptides between and within samples, the quantities determined from the characteristic peak areas being related to each other, wherein the contents determined by the characteristic peak areas are relative to each other, irrespective of the absolute weight to volume ratio or weight to weight content of the selected peptide in the protein preparation from the biological sample. The relative quantitative data on the individual characteristic peak areas between different samples was normalized with respect to the amount of protein analyzed for each sample. Relative quantification of a plurality of peptides from a plurality of proteins and one or more specified proteins, and/or relative quantification of a plurality of samples, can be performed simultaneously in a single sample to gain insight into the relative protein content, e.g., the content of one peptide/protein relative to other peptides/proteins.

Unlike SRM analysis, DIA does not require prior selection of target peptides. Instead, a precursor mass range is selected, and then the range is divided into a series of isolation windows. Mass spectra/mass spectra data were obtained from all detected precursor ions in the first isolation window. This operation is repeated for each successive adjacent isolation window until the entire precursor mass range is covered. Mass spectrometry/mass spectrometry libraries are used to identify peptides of interest from the acquired data, as described in more detail below.

Absolute quantitative levels of a given protein are determined by, for example, the SRM method, wherein the SRM characteristic peak area of an individual peptide from a given protein in a biological sample is compared to the SRM characteristic peak area of an incorporated internal standard. In one embodiment, the internal standard is a synthetic version of the same precise peptide derived from a given protein containing one or more amino acid residues labeled with one or more heavy isotopes. Such isotopically labeled internal standards are synthesized such that when analyzed by mass spectrometry, the standards produce a predictable and consistent characteristic peak of SRM that is distinct from the characteristic peak of the native peptide and can be used as a comparison peak. Thus, when the internal standard is incorporated into a protein preparation from a biological sample in a known amount and analyzed by mass spectrometry, the SRM characteristic peak area of the native peptide is compared to the SRM characteristic peak area of the internal standard peptide, and this numerical comparison indicates the absolute molar concentration and/or absolute weight of the native peptide present in the original protein preparation from the biological sample. Absolute quantitative data for fragment peptides are shown based on the amount of protein analyzed for each sample. Absolute quantification of multiple peptides and proteins can be performed in a single sample and/or simultaneously in multiple samples to gain insight into the absolute protein content in a single biological sample and in the entire population of single samples.

The assay methods described herein can be used to help diagnose the stage of cancer, e.g., directly in a patient-derived tissue, such as formalin-fixed tissue, and to help determine which therapeutic agent will be most beneficial for treating the patient. Cancerous tissue removed from a patient either surgically (e.g., to therapeutically remove a portion or the entire tumor) or by a biopsy procedure (which is performed to determine the presence or absence of a suspected disease) is analyzed to determine whether one or more specific proteins and what form of protein is present in the patient's tissue. In addition, the expression level of a protein or proteins can be determined and compared to a "normal" or reference level found in healthy tissue. The normal or reference level of protein found in healthy tissue can be derived, for example, from the relevant tissue of one or more individuals without cancer. Alternatively, by analyzing the relevant tissues not affected by cancer, normal or reference levels of cancer individuals can be obtained.

By using protein levels, determination of the protein level of one, some or all of the specified proteins can also be used to diagnose the stage of cancer in a patient or subject diagnosed with cancer. The absolute level of a single peptide derived from a given protein is defined as the molar amount of peptide determined by SRM assay/total amount of protein lysate analyzed. Thus, by correlating the level of a protein (or a fragment peptide from a protein) with the level observed in normal tissue, information about the specified protein can be used to help determine the stage or grade of cancer. Once the quantification of one or more specified proteins is determined in cancer cells, this information can be matched to a list of therapeutic agents (chemical and biological) developed for the specific treatment of cancer tissue characterized by, for example, the protein or abnormal expression of the protein being assayed. Matching the information from the protein assay to a list of therapeutic agents that specifically target, for example, a given protein or cell/tissue expressing the protein defines a so-called personalized medicine approach (personalized medicine) for treating a disease. The assay methods described herein form the basis for personalized medical procedures by using analysis of proteins from the patient's own tissues as a source of diagnostic and therapeutic decisions.

In principle, any predicted peptide derived from a given protein, e.g., prepared by digestion with a protease of known specificity (e.g., trypsin), can be used as an alternative reporter (reporter) to determine the abundance of the given protein in a sample using mass spectrometry-based SRM or DIA assays. Similarly, any predicted peptide sequence containing amino acid residues at sites known to be potentially modified in a given protein may also potentially be used to determine the degree of modification of a given protein in a sample.

Suitable fragment peptides derived from a given protein can be generated by a variety of methods, including by using the liquid tissue protocol provided in U.S. Pat. No. 7,473,532. in the liquid tissue protocol, tissues/organisms can be heated in a buffer (e.g., from about 80 ℃ to about 100 ℃ for a period of time from about 10 minutes to about 4 hours) for an extended period of time to reverse or release protein cross-links by proteolytic digestion of protein III.

Surprisingly, it has been found that many potential peptide sequences from the above proteins are not suitable or effective for mass spectrometry-based assays for reasons that are not yet apparent. Since it is not possible to predict the most suitable peptides for analysis, it is necessary to experimentally identify modified and unmodified peptides in actual liquid tissue lysates in order to develop a reliable and accurate analysis for each given protein. While not wishing to be bound by any theory, it is believed that some peptides may be difficult to detect, for example, by mass spectrometry because they do not ionize well or produce fragments that are different from other proteins. The peptides may also not dissolve well in the separation (e.g. liquid chromatography) or may adhere to glass or plastic vessels.

The peptides found in table 1 were derived from the respective indicated proteins by proteolytic digestion of all proteins in complex liquid lysates prepared from cells obtained from formalin-fixed cancer tissue. Unless otherwise indicated, the protease in each case was trypsin. The liquid tissue lysates are then analyzed by mass spectrometry to determine those peptides derived from the indicated proteins that were detected and analyzed by mass spectrometry. Identifying a particular preferred subset of peptide methods for mass spectrometry is based; 1) experimental determination of one or more peptides ionized from proteins in liquid tissue lysate mass spectrometry, and 2) the ability of the peptides to survive under the protocols and experimental conditions used to prepare the liquid tissue lysate. The latter property extends not only to the amino acid sequence of the peptide but also to the ability of modified amino acid residues in the peptide to survive in a modified form during sample preparation.

Protein lysates of cells obtained directly from formalin (formaldehyde) -fixed tissue were prepared using liquid tissue reagents and protocols that required collection of the cells into sample tubes by tissue microdissection followed by heating of the cells in liquid buffer for extended periods of time. Once formalin-induced cross-linking is negatively affected, the tissue/cells are then digested to completion in a predictable manner using proteases, including, but not limited to, the protease trypsin, for example. Each protein lysate was reduced to a collection of peptides by digestion of the intact polypeptide with a protease. Each liquid lysate is analyzed (e.g., by ion trap mass spectrometry) for multiple global proteomic studies of peptides, with data represented as many peptides as possible identified from all cellular proteins present in each protein lysate. Ion trap mass spectrometers or other forms of mass spectrometers capable of global analysis to identify as many peptides as possible from a single complex protein/peptide lysate are typically used. However, ion trap mass spectrometers may be the best type of mass spectrometer for performing global profiling (global profiling) of peptides.

Once as many peptides as possible have been identified in a single mass spectral analysis of a single lysate under the conditions employed, the check peptide list is then compiled and used to determine the proteins detected in that lysate.

An emerging approach, called Data Independent Acquisition (DIA), takes advantage of the power of both global proteomics and targeted approaches by combining the reproducibility of SRMs with the large number of proteins/peptides identified in global shotgun proteomics analysis. The DIA method generally avoids the need to detect individual precursor ions during analysis because the MS/MS scans are collected systematically (independently and without precursor information) throughout the acquisition process. A variety of DIA formats are in use and/or are currently being developed and any of the following may be employed in the methods described herein.

Data generation for DIA is more flexible and simpler than DDA or SRM experiments. The DIA acquires all MS/MS scans without regard to the selection of precursor ions from either the survey scan or the full MS scan, which is necessary for DDA. The predefinition of the target fragment peptide required for SRM/PRM is not necessary for DIA experiments. A wide range of precursors and corresponding transitions can be extracted after data acquisition. Thus, in targeted proteomics, the goal of DIA is to use targeted data extraction strategies for inclusive proteome-wide quantification. However, DIA-based targeting methods generally have lower sensitivity, specificity, and reproducibility, as well as a smaller dynamic range in protein quantification when compared to SRMs.

The method of D DIA was originally introduced using L TQ-Linear ion trap (L IT) mass spectrometers that applied a wide precursor separation window (10m/z) to perform sequential isolation and fragmentation of a predetermined m/z range.

Since then, other modified DIA have been introduced. Introducing MS on the basis of the above^EThe use of a smaller separation window (2.5U) results in improved protein identification, although overall data acquisition covering the entire target mass range requires multiple injections (67 injections for 5 days.) faster scanning ion trap MSs (e.g. L TQOrbitrapVelors MS) reduce the overall data acquisition time to about 2 days use the introduction of a bench top extraction Mass Spectrometry (a bench top exclusion MS) to demonstrate the application of whole ion fragmentation (AIF) in which peptides are injected into HCD collision cells for fragmentation without precursor selection and fragments are returned to the C-trap and analyzed by orbital trap massanalyzer (Orbitrap) this concept significantly reduces the duty cycle time but introduces more interference from another type of interference from the PTMF at some hold time this facilitates the distribution of fragment ions to co-eluting precursor ions with high mass precision this concept when compared to a unique ETIA mass spectrometer (PTMA) with a potential for increased mass spectrometric analysis based on the number of the extended spectrum of the peptide (about 250).

More recently, DIA has improved significantly with the development of fast scanning HR/AM instruments, whereby the change in DIA has been demonstrated using QqTOF MS, called SWATH, conceptually referring to the use of a wide isolation window consisting of multiple spectra (typically 25 m/z.) one key feature of using QqTOF MS is the fastest data acquisition rate Another DIA strategy has been introduced by using QqOrbi MS, where a new acquisition method called MSX has been introduced to improve instrument speed, selectivity and sensitivity. recently improvements in DIA data acquisition and processing have been aided by a newer version of Orbitrap MS instruments the first separation window, called Qqxctibit MS, was implemented on Qaxactionts MS, using an asymmetric separation window 5Da window coversing 400-800m/z over the mass range, 10Da window coversing 800m/z, 10Da 800m/z, 20Da, 10Da precursor ion trap 1000m/z, 20Da precursor ion trap with a continuous scanning ion masking (75-35) to cover the ion trap ion-ion trap with a continuous scanning of ion-ion detection and cross-ion detection, i.e. another method implemented using a continuous scanning ion masking (35-ion masking) with a wider ion masking window covering peak detection window, called SAIT-200-ion detection window, similar to obtain ion detection data, i.e. a simultaneous detection method with simultaneous detection of a continuous scanning of the detection of the ion masking, called DIA-ion masking, similar to the ion detection method, called DIA-200 ion masking, similar to the detection method, i.e. a continuous scanning ion masking, similar to the detection method, called DIA-200 ion masking, similar to the detection method, the detection.

Compared to standard DIA methods with complete recording of all fragment ions of a detectable peptide precursor but with complex data analysis, psmat and wiSIM-DIA can only provide a smaller number of MS/MS spectra for detectable precursors with relatively easier data analysis, since most of the duty cycle time is used to generate high mass MS1 data. Thus, their sensitivity and accuracy are higher than those provided by the standard DIA method, while their quantitative accuracy (i.e., specificity or selectivity) may be slightly lower than that of the standard DIA method, because MS1 has an increased chance of co-elution interference than MS/MS. Recently, a new DIA method called Hyper Reaction Monitoring (HRM) was demonstrated that includes integrated DIA acquisition on a Q exact MS platform and target data analysis with a retention time normalized (iRT) spectral library. HRM has been shown to be superior to global shotgun proteomics both in the number of consistently identified peptides and in the reliable quantification of different abundant proteins in multiple measurements.

Currently, there are three Methods for decoding DIA spectra, the first is to construct pseudo DDA spectra from DIA spectra, such as Demux, MaxQuant, XDIA processors and complementary finders, then process those reconstructed pseudo DDA spectra by conventional search engine tools, such as msmsgf +, MaxQuant, MASCOT or other spectral libraries, some schemes use chromatographic elution maps to improve peptide identification Tsou et al recently describe a development of a calculation method called DIA-Umpire, DIA-Umpire starts with a two-dimensional (m/z and retention time) feature detection algorithm to find all possible precursor and fragment ion signal in MS and MS/MS data, and ion fragment and precursor ion signal grouping 35ion groups are collected for the purpose of comprehensive peak processing with native ion peaks of native ion peaks, and natural ion peaks, see the above-related to MS peaks database, acquisition & metadata, map, the database for comprehensive analysis of MS and MS/MS peaks, and analysis Methods — the method includes the generation of precursor peaks for analysis of ion and protein peaks.

Another approach is to match multiple MS/MS to theoretical spectra of peptides (e.g., ProbIDtree, ion computing (IonAccounting), M-SP L IT, MixDB, and FT-ARM). Scoring algorithms are directly based on how many theoretical fragment ions of peptides from sequence databases or spectral libraries are found on multiple spectra with high mass accuracy.

Peptides detected

In one embodiment, the tryptic peptides identified as useful in determining the absolute or relative amount of a given protein are listed in table 1. Each of these peptides was detected by mass spectrometry in liquid lysates prepared from formalin fixed paraffin embedded tissues. Thus, each peptide can be used to develop a quantitative assay for a given protein in a human biological sample, including directly in formalin-fixed patient tissue.

TABLE 1

SEQ ID NO	Protein	Peptide sequences
			SEQ ID NO 1	CBL	LDLLPQR
SEQ ID NO 2	FPGS	SGLQVEDLDR
			SEQ ID NO 3	HSP90A	ALLFVPR
SEQ ID NO 4	HSP90B	ALLFIPR
			SEQ ID NO 5	INSR	TIDSVTSAQELR
SEQ ID NO 6	MCL1	LLFFAPTR
			SEQ ID NO 7	TrkA	WELGEGAFGK

The tryptic peptides listed in table 1 were typically detected from various liquid tissue lysates of various formalin-fixed tissues of various human organs, including, for example, the prostate, colon, and breast.

Although SRM analysis can be developed and performed on any type of mass spectrometer, including MA L DI, ion trap or triple quadrupole, the most advantageous instrument platform for SRM analysis is often considered the triple quadrupole instrument platform.

Measurement method

1. Identification of SRM candidate fragment peptides of proteins

a. Preparation of liquid tissue protein lysates from formalin-fixed biological samples using one or more proteases (which may or may not include trypsin) to digest proteins

b. Analyzing all protein fragments in a liquid tissue lysate on an ion trap tandem mass spectrometer and identifying all fragment peptides from a given protein, wherein a single fragment peptide does not comprise any peptide modification, such as phosphorylation or glycosylation

c. All protein fragments in liquid tissue lysates were analyzed on an ion trap tandem mass spectrometer and all fragment peptides were identified from proteins carrying peptide modifications (e.g., phosphorylated or glycosylated residues)

d. All peptides generated from full-length proteins by specific digestion methods are likely to be measured, but preferred peptides for developing SRM assays are those identified by mass spectrometry directly in complex liquid tissue protein lysates prepared from formalin-fixed biological samples

2. Mass spectrometric analysis of peptides of defined protein fragments

a. Single fragment peptides identified in liquid tissue lysates were subjected to SRM assays on a triple quadrupole mass spectrometer and applied to peptides from proteins

i. Determining the optimal retention time of the fragment peptide under optimal chromatographic conditions including, but not limited to, gel electrophoresis, liquid chromatography, capillary electrophoresis, nano-reversed phase liquid chromatography, high performance liquid chromatography, or reversed phase high performance liquid chromatography

Determining the monoisotopic mass of the peptides, the precursor charge state of each peptide, the precursor m/z value of each peptide, the m/z transition ion of each peptide, and the ion type of each transition ion of each fragment peptide, to develop an SRM assay for each peptide.

Then, the information from (i) and (ii) can be used to perform SRM assays on a triple quadrupole mass spectrometer, wherein each peptide has a characteristic and unique SRM characteristic peak that precisely defines the unique SRM assay performed on the triple quadrupole mass spectrometer

b. The SRM analysis is performed such that the content of fragment peptides of the protein being detected, as a function of the characteristic peak area of the unique SRM from the SRM mass spectrometry analysis, can be indicative of the relative and absolute amount of protein in a particular protein lysate.

i. Relative quantification can be achieved by:

1. determining an increase or decrease in protein by comparing the SRM characteristic peak area of a given fragment peptide detected in a liquid tissue lysate from one formalin-fixed biological sample with the same SRM characteristic peak area of the same fragment peptide in at least one second, third, fourth or more liquid tissue lysates from at least one second, third, fourth or more formalin-fixed biological samples

2. The increase or decrease in protein is determined by comparing the SRM characteristic peak area of a given fragment peptide detected from liquid tissue lysates of one formalin-fixed biological sample with the SRM characteristic peak areas generated from fragment peptides of other proteins, other samples obtained from different and isolated biological sources, wherein the SRM characteristic peak area comparison between two samples of peptide fragments is normalized to the amount of protein analyzed in each sample.

3. The increase or decrease in protein is determined by comparing the SRM characteristic peak area of a given fragment peptide to the SRM characteristic peak areas of other fragment peptides from different proteins in the same liquid tissue lysate from a formalin-fixed biological sample in order to normalize the altered level of the protein to the level of other proteins that do not alter their expression levels under various cellular conditions.

4. These assays are applicable to unmodified fragment peptides and modified fragment peptides of proteins, wherein modifications include, but are not limited to, phosphorylation and/or glycosylation, and wherein the relative levels of modified peptides are determined in the same manner as the relative amounts of unmodified peptides are determined.

Absolute quantitation of a given peptide can be achieved by comparing the SRM characteristic peak area of a given fragment peptide from a given protein in a single biological sample with the SRM characteristic peak area of an internal fragment peptide standard added to a biological sample protein lysate

1. Internal standards are labeled synthetic versions of the peptide of the specified protein fragment that were examined. Adding the standard substance into a sample with a known amount can determine the SRM characteristic peak areas of the internal fragment peptide standard substance and the natural fragment peptide in the biological sample respectively, and then comparing the two peak areas

2. This may apply to unmodified fragment peptides and modified fragment peptides, wherein modifications include, but are not limited to, phosphorylation and/or glycosylation, and wherein the absolute level of modified peptide may be determined in the same manner as the absolute level of unmodified peptide is determined.

3. Quantitative application of fragment peptides to cancer diagnosis and treatment

a. The relative and/or absolute quantification of the fragment peptide levels of a given protein and confirmation of the previously determined association of expression of a given protein with the stage/grade/status of cancer in a tumor tissue of a patient is well known in the cancer art

b. The relative and/or absolute quantification of the fragment peptide levels of a given protein and demonstration of correlation with clinical outcomes of different therapeutic strategies, where such correlation has been demonstrated in the field or can be demonstrated in the future by correlation studies across cohort patients and tissues from these patients. Once the previously established correlations or future derived correlations are confirmed by the analysis, the analysis method can be used to determine the optimal treatment strategy

By analyzing all fragment peptides on an ion trap and a triple quadrupole mass spectrometer, specificity and unique features were developed for the specific fragment peptide from each specified protein. The information for each and every candidate SRM peptide must be determined experimentally directly in liquid lysates from formalin-fixed samples/tissues; because, interestingly, not all peptides from any given protein could be detected in such lysates using the SRMs described herein, this indicates that the undetected fragment peptides cannot be considered as candidate peptides for the development of SRM assays for the direct quantification of peptides/proteins in liquid tissue lysates of formalin-fixed samples/tissues.

Specific SRM assays for specific fragment peptides were performed on a triple quadrupole mass spectrometer. The experimental samples analyzed by the specific protein SRM assay are for example liquid tissue protein lysates prepared from formalin-fixed and paraffin-embedded tissues. The data from such analysis indicated the presence of a distinct SRM signature peak for this fragment peptide in formalin fixed samples.

The specific transition ion characteristics of the peptide are used to quantitatively measure specific fragment peptides in formalin-fixed biological samples. These data indicate that the absolute amount of the fragment peptide is the molar amount of peptide per microgram of protein lysate analyzedAs a function of (c). Assessing the corresponding protein levels in tissues based on analysis of formalin-fixed patient-derived tissues can provide diagnostic, prognostic, and treatment-related information for each particular patient. In one embodiment, the present invention describes a method for measuring the level of each protein listed in table 1 in a biological sample comprising detecting and/or quantifying the amount of one or more modified or unmodified fragment peptides in a protein digest prepared from said biological sample using mass spectrometry; and calculating the level of modified or unmodified protein in the sample; and wherein the level is a relative level or an absolute level. In related embodiments, quantifying one or more fragment peptides comprises determining the amount of each fragment peptide in the biological sample by comparison to a known amount of an added internal standard peptide, wherein each fragment peptide in the biological sample is compared to an internal standard peptide having the same amino acid sequence. In some embodiments, the internal standard is an isotopically labeled internal standard peptide comprising one or more peptides selected from the group consisting of¹⁸O、¹⁷O、³⁴S、¹⁵N、¹³C、²A heavy stable isotope of H or a combination thereof.

The methods of the invention for measuring the level of a specified protein (or fragment peptide as a surrogate thereof) in a biological sample can be used as a diagnostic indicator of cancer in a patient or subject. In one embodiment, the results from the measurement of the level of a given protein may be used to determine the diagnostic stage/grade/status of a cancer by correlating (e.g., comparing) the level of protein found in a tissue with the level of protein found in normal and/or precancerous tissue.

Because both nucleic acids and proteins can be analyzed from the same liquid tissue biomolecule preparation, additional information about disease diagnosis and drug treatment decisions can be generated from nucleic acids in the same sample from which the protein is analyzed. For example, if a given protein is expressed by certain cells at increased levels, the data can provide information about the cell state and their likelihood of uncontrolled growth, potential drug resistance, and cancer development when determined by SRM. At the same time, information about the status of the corresponding genes and/or their encoded nucleic acids and proteins (e.g., mRNA molecules and their expression levels or splicing variations) can be obtained from nucleic acids present in the same liquid biomolecule preparation, which can be evaluated simultaneously with SRM analysis of a given protein. Any gene and/or nucleic acid that is not derived from the specified protein and is present in the same biomolecular product can be evaluated simultaneously with the SRM analysis of the specified protein. In one embodiment, information about a given protein and/or one, two, three, four or more additional proteins can be assessed by examining the nucleic acids encoding those proteins. These nucleic acids can be examined, for example, by one or more, two or more, or three or more of the following: sequencing methods, polymerase chain reaction methods, restriction fragment polymorphism analysis, identification of deletions, insertions and/or determination of the presence of mutations, including but not limited to single base pair polymorphisms, transitions, transversions or combinations thereof.

SEQUENCE LISTING

<110> Nantan Biomics, Limited liability company

<120> SRM and DIA assay for clinical cancer assessment

<130>I61053LRH

<150>62/791,495

<151>2019-01-11

<160>7

<170>PatentIn version 3.5

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Leu Asp Leu Leu Pro Gln Arg

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Ala Leu Leu Phe Val Pro Arg

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Ala Leu Leu Phe Ile Pro Arg

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Thr Ile Asp Ser Val Thr Ser Ala Gln Glu Leu Arg

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Claims

1. A method for measuring the amount of protein in a biological sample of formalin-fixed tissue, the method comprising:

detecting and/or quantifying the amount of one or more fragment peptides derived from said protein in a protein digest prepared from said biological sample using mass spectrometry; and calculating the amount of said protein in said sample;

wherein the protein is selected from the group consisting of CB L, FPGS, HSP90A, HSP90B, INSR, MC L1 and TrkA.

2. The method of claim 1, further comprising the step of fractionating said protein digest prior to detecting and/or quantifying said one or more fragment peptides.

3. The method of claim 2, wherein the step of fractionating is selected from the group consisting of gel electrophoresis, liquid chromatography, capillary electrophoresis, nano-reversed phase liquid chromatography, high performance liquid chromatography, and reversed phase high performance liquid chromatography.

4. The method of any of the preceding claims, wherein said protein digest comprises a protease digest, preferably a trypsin digest.

5. The method of any preceding claim, wherein the mass spectrometry comprises tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, ion trap/quadrupole hybrid mass spectrometry, MA L DI-TOF mass spectrometry, MA L DI mass spectrometry and/or time of flight mass spectrometry.

6. The method according to claim 5, wherein the mass spectrometric analysis mode used is Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM) and/or multiple selected reaction monitoring (mSRM).

7. The method according to claim 6, wherein the mode of mass spectrometry used is Data Independent Acquisition (DIA).

8. The method of any one of the preceding claims,

the protein is CB L, and the fragment peptide is a peptide shown as SEQ ID NO. 1;

the protein is FPGS, and the fragment peptide is SEQ ID NO: 2;

the protein is HSP90A, and the fragment peptide is SEQ ID NO: 3;

the protein is HSP90B, and the fragment peptide is SEQ ID NO: 4;

the protein is INSR and the fragment peptide is SEQ ID NO: 5;

the protein is MC L1, the fragment peptide is a peptide shown as SEQ ID NO. 6, and/or

The protein is TrkA, and the fragment peptide is SEQ ID NO: 7.

9. The method of any one of the preceding claims, wherein the tissue is paraffin embedded tissue.

10. The method of any one of the preceding claims, wherein the tissue is obtained from a tumor, e.g., a primary tumor or a secondary tumor.

11. The method of any one of the preceding claims, wherein quantifying the one or more fragment peptides comprises comparing the amount of the one or more fragment peptides in the biological sample to the amount of the same one or more fragment peptides in a different and separate biological sample.

12. The method of any one of the preceding claims, wherein quantifying the one or more fragment peptides comprises determining the amount of the one or more fragment peptides in the biological sample by comparison to an added known amount of an internal standard peptide having the same amino acid sequence.

13. The method of claim 12, wherein the internal standard peptide is an isotopically labeled peptide selected from the group consisting of¹⁸O、¹⁷O、³⁴S、¹⁵N、¹³C and²h and combinations thereof.

14. The method of any of the preceding claims, wherein detecting and/or quantifying the amount of the one or more fragment peptides in the protein digest indicates the presence of the corresponding protein and an association with the diagnostic stage/grade/status of cancer in the subject.

15. The method of claim 14, further comprising correlating the results of said detecting and/or quantifying the amount of said one or more fragment peptides or the amount of said corresponding protein to the optimal cancer treatment therapy for said subject.

16. The method of claim 15, wherein the correlation is combined with detecting and/or quantifying the content of other proteins or peptides from other proteins in a multiplex format.