# Patent

## Title
Secretome profile-facilitated in vitro fertilization

## Abstract

Secretome profiling improves the pregnancy success rate of in vitro fertilization processes, while reducing the risk of multiple births.

## Background
RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 15/052,475, filed Feb. 24, 2016, issued on Jul. 25, 2017 as U.S. Pat. No. 9,714,950, which is a continuation of U.S. application Ser. No. 13/081,463, filed Apr. 6, 2011, which claims benefit of priority to U.S. Provisional Patent Application No. 61/321,448 filed Apr. 6, 2010, each of which is incorporated by reference to the same extent as though fully replicated herein.


SEQUENCE LISTING
This application includes a Sequence Listing, as set forth in an ASCII-compliant text file named “CCRMProtein_ST25.txt”, created on Apr. 6, 2011, and containing 2263 kilobytes, which is incorporated by reference to the same extent as though fully replicated herein.
BACKGROUND
Field
The invention relates to the field of in vitro fertilization (IVF), which is a process by which mammalian egg cells are fertilized by sperm outside the womb. More particularly, evaluation of a secretome profile is used to enhance the pregnancy success rate when the fertilized egg is implanted into a patient's uterus.
Description of the Related Art
IVF infertility treatment offers infertile couples a chance to have a biologically related child. IVF may overcome female infertility due to problems of the fallopian tube or endometriosis. IVF also overcomes male infertility due to problems with sperm quality or quantity. The IVF process involves hormonally controlling the ovulatory process, removing eggs (termed ova) from the woman's ovaries and permitting the sperm to fertilize the eggs in a fluid medium. The fertilized egg, termed an embryo, is subsequently transferred to the patient's uterus with the intent of establishing a successful pregnancy. Due to expensive procedural costs, IVF is only attempted after the failure of less expensive fertility treatments.
FIG. 1 displays a schematic for mammalian blastocyst implantation. Mammalian embryo implantation is a complex and intricate process involving numerous biological changes at both the embryo and endometrial level. The interaction between the blastocyst and the endometrium is a function of both a receptive endometrial environment and a healthy blastocyst. A blastocyst failing to implant or an endometrium failing to sustain growth and differentiation will result in spontaneous abortion. The prior art teaches very little about the embryo's role in the events leading to the attachment of a viable blastocyst to a receptive uterine luminal epithelium.
IVF treatment begins with administration of hormonal medications to stimulate ovarian follicle production, such as gonadotropins hormones. The prevention of spontaneous ovulation involves using other hormones, such as GnRH antagonists or GnRH agonists that block the natural surge of luteinizing hormone. With adequate follicular maturation, administration of human chorionic gonadotropin hormone causes ovulation approximately 42 hours after the administration. However, the egg retrieval procedure takes place just prior to ovulation, in order to recover the eggs from the ovary. The egg retrieval proceeds using a transvaginal technique involving an ultrasound-guided needle that pierces the vaginal wall to reach the ovaries. After recovery of the follicles through the needle, the follicular fluid is provided to the IVF laboratory to identify eggs. Typically, the procedure retrieves between 10 and 30 eggs. The retrieval procedure takes approximately 20 minutes and is usually done under conscious sedation or general anesthesia.
For IVF, the fertilization of the egg (termed insemination) proceeds in the laboratory where the identified eggs and semen are usually incubated together in a culture media. The confirmation of fertilization proceeds by monitoring the eggs for cell division. For instance, a fertilized egg may show two pronuclei. In certain situations, such as low sperm count or motility, a single sperm may be injected directly into the egg using a method called intracytoplasmic sperm injection (ICSI). In another option known as gamete intrafallopian transfer, eggs are removed from the woman and placed in one of the fallopian tubes, along with the man's sperm. In this example, fertilization occurs within the women's body, a process termed in vivo fertilization.
Selected embryos are transferred to the patient's uterus through a thin, plastic catheter, which goes through the vagina and cervix. Typically, transfer of 6-8 cell stage embryos to the uterus occurs three days after embryo retrieval. Alternatively, embryos can be placed into an extended culture system with a transfer done at the blastocyst stage at approximately five days post-retrieval. Blastocyst stage transfers often result in higher pregnancy rates. Additionally, embryonic cryopreservation, or the storage of embryos in a frozen state, is feasible until uterine transfer. For example, the first term pregnancy derived from a frozen human embryo was reported in 1984.
Despite progressively improving IVF pregnancy rates, the majority of transferred human embryos result in implantation failure. For example, Canadian clinics reported an average pregnancy rate of 35% for one cycle, but a live birth rate of only 27% in 2006. Moreover, implantation success rates may decrease with the increasing maternal age, if donor eggs are not used. Various factors are associated with implantation failure, including embryo chromosome aneuploidies related to advanced maternal age and maternal factors such as endometrium response failure to hormone regulation.
To overcome low implantation success rate, multiple embryos are commonly transferred during a single IVF procedure. The process for selecting embryos for transfer often involves grading methods developed in individual laboratories to judge oocyte and embryo quality. An arbitrary embryo score, involving the number and quality of embryos, may reveal the probability of pregnancy success post-transfer. For example, an embryologist may grade embryos using morphological qualities including the number of cells, clearness of cytoplasm, evenness of growth and degree of fragmentation. However, embryo selection based on morphological qualities is not precise. Often, several embryos selected for these general qualities are implanted to improve the chance of pregnancy. The number of embryos transferred depends upon the number available, the age of the woman and other health and diagnostic factors.
The transfer of multiple embryos, however, often results in multiple pregnancies, a major complication of IVF. In general, multiple pregnancies, specifically, more than twins, hold maternal and fetal risks. For example, multiple births are associated with increased risk of pregnancy loss, neonatal morbidity, obstetrical complications, and prematurity with potential for long term damage. Some countries implemented strict limits on the number of transferred embryos to reduce the risk of high-order multiples (e.g., triplets or more). However, these limitations are not universally followed or accepted.
SUMMARY
In one embodiment, a system for enhancing the pregnancy success rate of in vitro fertilization includes a means for determining the secretome profile of an embryo to identify proteins, polypeptides, oligopeptides or protein fragments implicated in implantation success and a means for recommending whether to implant the embryo, such as a blastocyst, on the basis of the secretome profile.
The CDC data from 2006 shows that use of donor eggs in a 40 year old woman will result in a live birth 54% of the time. This compares to 20.6% using the woman's own eggs. Thus, an opportunity exists for the instrumentalities of this disclosure to facilitate success rates approximating the use of donor eggs when using one's own eggs, and may even permit higher success rates due to better selection of viable embryos.
In an embodiment, a system is provided for enhancing the pregnancy success rate of in vitro fertilization. The system includes an electronic system to configured to gather secretome data as a secretome profile of an embryo by quantitating proteins implicated in implantation success. A model is provided for use in recommending whether to implant the embryo on the basis of this secretome profile. The secretome date may be, for example, provided by use of mass spectroscopy or ELISA measurements.
According to one aspect of the system, the secretome profile may be provided by using proteins as embryo secretions in culture media that may be linked to changed odds of implantation success, for example, as may be found in one or more sequences found in SEQID Nos. 1-404. The sequences of SEQID Nos. 261-404 are preferred for this use. Particularly preferred are the sequences of SEQID Nos. 310, 311, 313, 317, 318, 319, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 383, 391, 397, 398, 399, 402, 403, and 404. The sequences of SEQID Nos. 310, 311, 313, 318, 319, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 391, 397, 398, 399, 402, 403, and 404 are associated with increased odds of implantation success, whereas those of SEQID Nos. 317 and 383 are associated with failure or aneuploidy.
In an embodiment, a method of in vitro fertilization entails determining the secretome profile of a candidate embryo by use of these sequences to ascertain proteins implicated in implantation success. This provides data that may be submitted to a model that associates one or more of these proteins with changes odds of implantation success or failure. A recommendation for implantation of the embryo may then be provided based upon the modeling outcome. The embryo may be conditionally implanted on the basis of the recommendation.
In an embodiment, there is an improved ELISA assay kit with a plurality of microwells for the quantitation of protein content in a sample. The microwells are constructed and arranged to quantitate for a plurality of proteins selected from these sequences.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a diagram illustrating the process blastocyst implantation.
FIG. 2 is a table that illustrates the success rates from donor oocyte IVF cycles.
FIG. 3 is a flow chart for a method of developing and using a predictive model that may be used to assess embryo viability as a candidate for use in an IVF implantation procedure.
FIG. 4 shows a system that analyzes the secretome of an embryo to assess viability.



DETAILED DESCRIPTION
The following definitions are provided to facilitate understanding of certain terms used herein and are not meant to limit the scope of the present disclosure.
The term “secretome panel” refers to a collection of individual proteins, polypeptides, oligopeptides or protein fragments that are differentially expressed and secreted by an embryo. The proteins, polypeptides, oligopeptides or protein fragments of the secretome panel are selected based on predictions of developmental competence and implantation potential of an embryo.
The term “spent media” refers to media surrounding an embryo that accumulates proteins secreted from the embryo.
The term “secretome profiling” refers to qualitative and quantitative analysis of a secretome panel collected from spent media.
There will now be shown and described a system for enhancing the pregnancy success rate of in vitro fertilization involving non-invasive secretome profiling of an embryo. Secretome profiling may be performed on embryos at differing embryonic developmental stages, such as day one through day six embryos.
Non-limiting examples of proteins comprising the secretome panel include proteins involved in amino acid metabolism, lipid metabolism, carbohydrate metabolism, signal transduction, apoptosis, transcription and combinations thereof. In one example, an embryo secretes a protein that is involved in amino acid metabolism, thereby indicating potential for successful embryo implantation. In another example, an embryo secretes a protein that is involved in apoptosis, thereby indicating a reduced potential for successful blastocyst implantation. Nonlimiting examples of the number of proteins comprising the secretome panel includes ≧250 proteins, ≧100 proteins, ≧50 proteins, ≧20 proteins or ≧10 proteins.
Secretome profiling involves assessment of a secretome panel. Secretome profiling is a noninvasive method for predicting developmental competence and viability of an embryo. Comparison of secretome profiles between developing embryos and degenerating embryos, both at the same developmental stage, reveals significant differences in protein expression. Moreover, secretome profiling provides a molecular perspective of the functioning biochemical pathways present during embryo development. A noninvasive secretome profiling assay correlates embryonic secretome to embryonic viability, thereby facilitating single embryo transfer during in vitro fertilization.
FIG. 2 highlights the importance of embryo developmental competence by showing high success rates from donor oocyte IVF cycles using young reproductive age oocytes transferred to an advanced maternal age endometrium. The data is provided for a Colorado clinic from 2004-2009 including outcomes for over 1000 donor oocyte cycles. These results demonstrate a 66.6% implantation rate for IVF recipients with an average endometrial age of 40.6 years and donor oocytes with an average age of 26.6 years.
In one embodiment, secretome profiling occurs by assessing a secretome panel via Enzyme-Linked Immunosorbent Assay (ELISA). In another embodiment, secretome profiling proceeds by assessing a secretome panel via mass spectrometry.
Non-limiting examples of solid-state substrates used in ELISA include microwell plates, such as 96-well plates, 384-well plates and 8-well strips, microarray slides and nitrocellulose membranes. In one embodiment, glass microarray surfaces contain chemical functional groups such as epoxy, amine or aldehyde. In one embodiment, microwell plate material comprises polystyrene.
The term “capture antibody” refers to an antibody secured, either covalently or non-covalently, to a solid-state substrate. The capture antibody recognizes and binds to a specific antigen such as a protein, a polypeptide, an oligopeptide, a protein fragment, a carbohydrate or a small molecule. In one example, a solid-state substrate is functionalized with capture antibodies by passive adsorption or by specific binding. For example, specific binding of capture antibodies may occur using biotinylated capture antibodies and streptavidin-coated solid-state substrates. In another example, solid-state substrates are functionalized with antigens via passive adsorption.
Non-limiting examples of ELISA types include direct, indirect, competitive, and sandwich. In one example, proteins from the secretome panel are detected on a solid-state substrate using either a primary labeled antibody or a secondary labeled antibody. In another example, proteins from the secretome panels are detecting using a sandwich ELISA wherein a protein from the secretome panel is bound between two primary antibodies, namely the capture antibody and the detection antibody. Non-limiting examples of antibody labels include enzymes and fluororphores. In one embodiment, an enzyme that is conjugated to a detection antibody binds to a substrate producing either a chromogenic, fluorescent or chemiluminescent signal that is proportional to the quantity of protein from the secretome panel.
FIG. 3 shows a method 300 implementing the concepts described above to identify those embryos having the greatest chanced for implantation success rates. Step 302 entails ascertaining the secretome of proteins secreted into culture medium by an egg, blastocyst or developing embryo (hereinafter “embryo”) that has been previously fertilized by methods known to the art. Step 302 may be performed at any time, including before an egg is fertilized or immediately thereafter. More typical times to sample the media include form one to five days after fertilization occurs. Step 302 is preferably conducted before step 304, but samples of the culture medium may be frozen for later analysis. The embryo is then used 304 in an IVF procedure attempting to implant the embryo in a woman who desires to have a baby. The implantation successes and failures are monitored 306 to collect data relating the successes and failures to one or more proteins of the secretome. Once a sufficient number of instances have transpired 308 to provide for statistical significance, the proteins are mapped 310 to the instances of success and failure. The exact manner of mapping is not of particular importance, as there a number of biostatistical analysis software products are commercially available. For example, this may be done using the JMP® 9 packages from SAS of Cary, N.C. to implement methods as taught by Zar, Biostatistical Analysis, 5th Ed., Prentice Hall (2007) to map the secretome profiles using also time as a variable. The mapping may be multivariate, or by an artificially intelligent algorithm, such as a neural network. This model may be then used 312 as a predictive tool to select embryos for implantation based upon their secretome profile. This is done by analyzing 314 the secretome of candidate embryos for implantation with use of the model to predict implantation success or failure. One or more of the selected embryos are then implanted 316
FIG. 4 shows an analytical system 400 that may be used in step 314 according to one embodiment. A specimen 402 contains a sample of culture medium that is collected for secretome analysis as described above. By way of example, this specimen may contain culture medium in the form of a single or multi-well ELISA assay, or in a form that has been pre-processed for mass spectroscopy analysis. A corresponding measurement device 404 obtains primary measurements from the specimen 402. The measurements are representative of one or more proteins in the secretome that have been mapped to incidences of implantation success of failure. The model for this mapping is provided in step 312 of method 300 and resides on analyzer 406, which may be a computer operating on program instructions. The analyzer 406 receives signals from the measurement device 404 and interprets these by computational analysis according to the model then provides the results of computational analysis to an output device 408, which may be printer or an optical display. The various components of system 400 may be partially integrated, such as by combining the measurement device 404, analyzer 406, and output device 408 into one integrated system.
It will be appreciated that perceptive use of the instrumentalities described herein may result in a better selection of healthy embryos, such as blastocysts, for implantation. Thus, fewer blastocysts need to be implemented, such that there is lower risk of multiple pregnancies while achieving a higher overall pregnancy success rate.
The following descriptions will show and describe, by way of non-limiting examples, a process for improving pregnancy success rates with lower incidence of multiple births. The following examples describe secretome profiling of spent media from human blastocysts to provide implantation recommendation. It is to be understood that these examples are provided by way of illustration and should not be unduly construed to limit the scope of what is disclosed herein.
Example 1
Secretome Profiling of Human Embryos Using Mass Spectrometry
The following nonlimiting example teaches by way of illustration, not by limitation, secretome profiling of a human embryo using mass spectrometry (MS). Human cleavage-stage embryos were cultured in 10 μL drops of G1 supplemented with 2.5 mg/mL recombinant albumin under oil at 37° C., 6% CO2, 5% O2 for 24 hours. The embryos were washed twice in G2 culture media and further cultured in 10 μL drops of G2 supplemented with 2.5 mg/mL recombinant albumin under oil at 37° C., 6% CO2, 5% O2 for 48 hours with a fresh drop of G2 media added after 24 hours. Spent media samples of blastocysts were transferred into 0.65 mL Eppendorf tubes. Control groups comprised media cultured and collected under the same conditions but without embryos.
Micro-drops of spent media were depleted of human serum albumin (HSA) using Cibracron Blue Activated SwellGel Discs (Themo Fisher Scientific, Rockford, Ill.). The proteins in the spent media were separated by 1D gel electrophoresis (Invitrogen, Carlsbad, Calif.), followed by Coomassie staining. Twenty five individual bands were cut out from each sample lane and a standard in-gel digestion protocol was used based on previously used methods ((1.) Rosenfeld, J., Capdevielle, J., Guillemot, J. C. & Ferrara, P. In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal. Biochem. 203, 173-179 (1992), (2.) Hellman, U., Wernstedt, C., Gonez, J. & Heldin, C. H. Improvement of an “In-Gel” digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal. Biochem. 224, 451-455 (1995)). Iodoacteamide (IAM) was used for cysteine alkylation. The samples were analyzed on a LTQ-FT Ultra Hybrid Mass Spectrometer (Thermo/Finnigan; Waltham, Mass.) with a method based on a previously described protocol from Hansen et al., (Hansen KC, Kiemele L, Mailer O, O'Brien J, Shankar A, Fornetti J, Schedin P. An in-solution ultrasonication-assisted digestion method for improved extracellular matrix proteome coverage. Mol Cell Proteomics. 8(7):1648-57 (2009)).
Spent media samples were analyzed on a LTQ-FT Ultra Hybrid Mass Spectrometer (Thermo/Finnigan; Waltham, Mass.). Peptide desalting and separation was achieved using a dual capillary/nano pump HPLC system (Agilent 1200, Palo Alto, Calif.). On this system 8 μL of spent media sample was loaded onto a trapping column (ZORBAX 300SB-C18, dimensions 5×0.3 mm 5 um) and washed with 5% acetonitrile (ACN), 0.1% formic acid (FA) at a flow rate of 15 μL/min for 5 minutes. At this time, the trapping column was put online with the nano-pump at a flow rate of 350 nL/min. An 85 minute gradient, from 8% ACN to 40% ACN, was used to separate the peptides. The column was made from an in-house pulled 360/100 nm (outer/inner diameter) fused silica capillary packed with Jupiter C18 resin (Penomenex; Torrance, Calif.). The column was kept at a constant 40° C. using an in-house built column heater. Data acquisition was performed using the instrument supplied Xcalibur (version 2.0.6) software. The LC runs were monitored in positive ion mode by sequentially recording survey MS scans (m/z 400-2000), in the ICR cell, while three MS2 were obtained in the ion trap via CID for the most intense ions. After two acquisitions of a given ion within 45 seconds, the ion was excluded for 150 seconds.
For data analysis, The Raw Distiller program (UCSF) was used to create a de-isotoped centroided peak lists from the raw spectra into the mascot format using the default settings. The peak lists were searched against the SwissProt Human database (51.6, Homo sapiens 15720 sequences) using MascotTM server (Version 2.2, Matrix Science, Boston, Mass.). The search parameters are the same as those followed in Hansen et al., (Hansen K C, Kiemele L, Maller O, O'Brien J, Shankar A, Fornetti J, Schedin P. An in-solution ultrasonication-assisted digestion method for improved extracellular matrix proteome coverage. Mol Cell Proteomic. 8(7): 1648-57 (2009)). The Mascot results were loaded into Scaffold (v 2.06) and the runs were compared.
In one embodiment, MS analysis of spent media reveals a secretome panel of 261 individual proteins (Table 1). In one example, secretome profiling using the secretome panel of 261 proteins (Table 1), as analyzed by MS, identifies the potential for developmental competence and implantation success of a human embryo.
In one embodiment, analysis of spent media via MS reveals a secretome panel of 37 individual proteins (Table 2). Secretome profiling via MS of 37 individual proteins (Table 2) from spent media of human embryos correlates with embryonic viability and euploidy. The term “euploidy” refers to having a chromosome number that is an exact multiple of the haploid number for a human embryo, namely 23 pairs of chromosomes.
In Tables 1 and 2 below, the entries for “Entry Name” and ““Accession Number” refer to identifiers for published sequence data that is stored in bioinformatic databases including the Uniprot Knowledgebase, Swiss-Prot and TrEMBL. The information is made freely available to the world and is coordinated by the Swiss Institute of Bioinformatics, which is centrally administered in Lausanne, Switzerland with offices in Bern, Geneva and Zurich. The sequence information represented by these identifiers together with the representative publications is hereby incorporated by reference to the same extent as though fully replicated herein. For those proteins having isoforms, the sequences for Table 1 include consensus sequences for the primary isoforms, while sequences for Table 2 include also all other isoforms available at the time of filing.







TABLE 1







Proteins identified in spent media of human embryos.












SEQ


Protein




ID


Molecular
Accession



NO.
Protein Name
Entry Name
Weight
Number
Taxonomy
















1
Serum albumin
ALBU_HUMAN
69
kDa
P02768
Homo sapiens


2
Serotransferrin
TRFE_HUMAN
77
kDa
P02787
Homo sapiens


3
Haptoglobin
HPT_HUMAN
45
kDa
P00738
Homo sapiens


4
Alpha-1-antitrypsin
A1AT_HUMAN
47
kDa
P01009
Homo sapiens


5
Alpha-2-macroglobulin
A2MG_HUMAN
163
kDa
P01023
Homo sapiens


6
Hemopexin
HEMO_HUMAN
52
kDa
P02790
Homo sapiens


7
Apolipoprotein A-I
APOA1_HUMAN
31
kDa
P02647
Homo sapiens


8
Ceruloplasmin
CERU_HUMAN
122
kDa
P00450
Homo sapiens


9
Vitamin D-binding protein
VTDB_HUMAN
53
kDa
P02774
Homo sapiens


10
Alpha-1-antichymotrypsin
AACT_HUMAN
48
kDa
P01011
Homo sapiens


11
Plasma protease C1 inhibitor
IC1_HUMAN
55
kDa
P05155
Homo sapiens


12
Alpha-1B-glycoprotein
A1BG_HUMAN
54
kDa
P04217
Homo sapiens


13
Hemoglobin subunit beta
HBB_HUMAN
16
kDa
P68871
Homo sapiens


14
Antithrombin-III
ANT3_HUMAN
53
kDa
P01008
Homo sapiens


15
Afamin
AFAM_HUMAN
69
kDa
P43652
Homo sapiens


16
Angiotensinogen
ANGT_HUMAN
53
kDa
P01019
Homo sapiens


17
Transthyretin
TTHY_HUMAN
16
kDa
P02766
Homo sapiens


18
Inter-alpha-trypsin inhibitor heavy
ITIH4_HUMAN
103
kDa
Q14624
Homo sapiens



chain H4







19
Complement factor B
CFAB_HUMAN
86
kDa
P00751
Homo sapiens


20
Hemoglobin subunit alpha
HBA_HUMAN
15
kDa
P69905
Homo sapiens


21
Ig alpha-1 chain C region
IGHA1_HUMAN
38
kDa
P01876
Homo sapiens


22
Ig gamma-1 chain C region
IGHG1_HUMAN
36
kDa
P01857
Homo sapiens


23
Heparin cofactor 2
HEP2_HUMAN
57
kDa
P05546
Homo sapiens


24
Ig gamma-2 chain C region
IGHG2_HUMAN
36
kDa
P01859
Homo sapiens


25
Ig kappa chain C region
KAC_HUMAN
12
kDa
P01834
Homo sapiens


26
N-acetylmuramoyl-L-alanine
PGRP2_HUMAN
62
kDa
Q96PD5
Homo sapiens



amidase







27
Alpha-2-HS-glycoprotein
FETUA_HUMAN
39
kDa
P02765
Homo sapiens


28
Kininogen-1
KNG1_HUMAN
72
kDa
P01042
Homo sapiens


29
Zinc-alpha-2-glycoprotein
ZA2G_HUMAN
34
kDa
P25311
Homo sapiens


30
Thyroxine-binding globulin
THBG_HUMAN
46
kDa
P05543
Homo sapiens


31
Corticosteroid-binding globulin
CBG_HUMAN
45
kDa
P08185
Homo sapiens


32
Attractin
ATRN_HUMAN
159
kDa
O75882
Homo sapiens


33
Pregnancy zone protein
PZP_HUMAN
164
kDa
P20742
Homo sapiens


34
Alpha-2-antiplasmin
A2AP_HUMAN
55
kDa
P08697
Homo sapiens


35
Vitronectin
VTNC_HUMAN
54
kDa
P04004
Homo sapiens


36
Ig lambda chain C regions
LAC_HUMAN
11
kDa
P01842
Homo sapiens


37
Apolipoprotein A-II
APOA2_HUMAN
11
kDa
P02652
Homo sapiens


38
Apolipoprotein C-III
APOC3_HUMAN
11
kDa
P02656
Homo sapiens


39
Insulin-like growth factor-binding
ALS_HUMAN
66
kDa
P35858
Homo sapiens



protein complex acid labile chain







40
Complement C3
CO3_HUMAN
187
kDa
P01024
Homo sapiens


41
Complement C4-B
CO4B_HUMAN
193
kDa
P0C0L5
Homo sapiens


42
Serum paraoxonase/arylesterase 1
PON1_HUMAN
40
kDa
P27169
Homo sapiens


43
Plasma retinol-binding protein
RETBP_HUMAN
23
kDa
P02753
Homo sapiens


44
Carboxypeptidase N subunit 2
CPN2_HUMAN
61
kDa
P22792
Homo sapiens


45
Beta-2-glycoprotein 1
APOH_HUMAN
38
kDa
P02749
Homo sapiens


46
Inter-alpha-trypsin inhibitor heavy
ITIH2_HUMAN
106
kDa
P19823
Homo sapiens



chain H2







47
Clusterin
CLUS_HUMAN
52
kDa
P10909
Homo sapiens


48
Protein AMBP
AMBP_HUMAN
39
kDa
P02760
Homo sapiens


49
Ig heavy chain V-III region BRO
HV305_HUMAN
13
kDa
P01766
Homo sapiens


50
Dynactin subunit 2
DCTN2_HUMAN
44
kDa
Q13561
Homo sapiens


51
Inter-alpha-trypsin inhibitor heavy
ITIH1_HUMAN
101
kDa
P19827
Homo sapiens



chain H1







52
Complement C2
CO2_HUMAN
83
kDa
P06681
Homo sapiens


53
Serum amyloid P-component
SAMP_HUMAN
25
kDa
P02743
Homo sapiens


54
Lamin-A/C
LMNA_HUMAN
74
kDa
P02545
Homo sapiens


55
Phosphatidylinositol-glycan-specific
PHLD1_HUMAN
92
kDa
P80108
Homo sapiens



phospholipase D







56
Alpha-1-acid glycoprotein 1
A1AG1_HUMAN
24
kDa
P02763
Homo sapiens


57
Pigment epithelium-derived factor
PEDF_HUMAN
46
kDa
P36955
Homo sapiens


58
Apolipoprotein C-I
APOC1_HUMAN
9
kDa
P02654
Homo sapiens


59
Transcription intermediary factor 1-
TIF1B_HUMAN
89
kDa
Q13263
Homo sapiens



beta







60
Leucine-rich alpha-2-glycoprotein
A2GL_HUMAN
38
kDa
P02750
Homo sapiens


61
Lumican
LUM_HUMAN
38
kDa
P51884
Homo sapiens


62
Ig kappa chain V-III region SIE
KV302_HUMAN
12
kDa
P01620
Homo sapiens


63
Actin, cytoplasmic 1
ACTB_HUMAN
42
kDa
P60709
Homo sapiens


64
Apolipoprotein E
APOE_HUMAN
36
kDa
P02649
Homo sapiens


65
Kallistatin
KAIN_HUMAN
49
kDa
P29622
Homo sapiens


66
Myosin-15
MYH15_HUMAN
225
kDa
Q9Y2K3
Homo sapiens


67
Carboxypeptidase B2
CBPB2_HUMAN
48
kDa
Q96IY4
Homo sapiens


68
Serum amyloid A-4 protein
SAA4_HUMAN
15
kDa
P35542
Homo sapiens


69
Cholinesterase
CHLE_HUMAN
68
kDa
P06276
Homo sapiens


70
Apolipoprotein D
APOD_HUMAN
21
kDa
P05090
Homo sapiens


71
Tubulin beta-2C chain
TBB2C_HUMAN
50
kDa
P68371
Homo sapiens


72
Haptoglobin-related protein
HPTR_HUMAN
39
kDa
P00739
Homo sapiens


73
Hemoglobin subunit delta
HBD_HUMAN
16
kDa
P02042
Homo sapiens


74
Ig kappa chain V-IV region Len
KV402_HUMAN
13
kDa
P01625
Homo sapiens


75
Ig alpha-2 chain C region
IGHA2_HUMAN
37
kDa
P01877
Homo sapiens


76
Proteoglycan 4
PRG4_HUMAN
151
kDa
Q92954
Homo sapiens


77
Histone H2A type 1-D
H2A1D_HUMAN
14
kDa
P20671
Homo sapiens


78
Biotinidase
BTD_HUMAN
61
kDa
P43251
Homo sapiens


79
Carboxypeptidase N catalytic chain
CBPN_HUMAN
52
kDa
P15169
Homo sapiens


80
Plasma kallikrein
KLKB1_HUMAN
71
kDa
P03952
Homo sapiens


81
Ig kappa chain V-II region TEW
KV204_HUMAN
12
kDa
P01617
Homo sapiens


82
Apolipoprotein C-II
APOC2_HUMAN
11
kDa
P02655
Homo sapiens


83
DNA-dependent protein kinase
PRKDC_HUMAN
469
kDa
P78527
Homo sapiens



catalytic subunit







84
Heterogeneous nuclear
HNRL1_HUMAN
96
kDa
Q9BUJ2
Homo sapiens



ribonucleoprotein U-like protein 1







85
Apolipoprotein B-100
APOB_HUMAN
516
kDa
P04114
Homo sapiens


86
Heterogeneous nuclear
ROA2_HUMAN
37
kDa
P22626
Homo sapiens



ribonucleoproteins A2/B1







87
Tubulin alpha-ubiquitous chain
TBAK_HUMAN
50
kDa
P68363
Homo sapiens


88
Gelsolin
GELS_HUMAN
86
kDa
P06396
Homo sapiens


89
Tetranectin
TETN_HUMAN
23
kDa
P05452
Homo sapiens


90
Alpha-1-acid glycoprotein 2
A1AG2_HUMAN
24
kDa
P19652
Homo sapiens


91
Ig heavy chain V-III region GAL
HV320_HUMAN
13
kDa
P01781
Homo sapiens


92
IgGFc-binding protein
FCGBP_HUMAN
572
kDa
Q9Y6R7
Homo sapiens


93
Lactotransferrin
TRFL_HUMAN
78
kDa
P02788
Homo sapiens


94
Histone H4
H4_HUMAN
11
kDa
P62805
Homo sapiens


95
Glutathione peroxidase 3
GPX3_HUMAN
26
kDa
P22352
Homo sapiens


96
Histone H2B type 1-M
H2B1M_HUMAN
14
kDa
Q99879
Homo sapiens


97
Apolipoprotein M
APOM_HUMAN
21
kDa
Q95445
Homo sapiens


98
E3 SUMO-protein ligase RanBP2
RBP2_HUMAN
358
kDa
P49792
Homo sapiens


99
Ig kappa chain V-I region EU
KV106_HUMAN
12
kDa
P01598
Homo sapiens


100
Ig kappa chain V-I region Lay
KV113_HUMAN
12
kDa
P01605
Homo sapiens


101
Dermcidin OS = Homo sapiens
DCD_HUMAN
11
kDa
P81605
Homo sapiens


102
Leucine-rich repeats and
LRIG2_HUMAN
119
kDa
O94898
Homo sapiens



immunoglobulin-like domains








protein 2







103
Protein S100-A8
S10A8_HUMAN
11
kDa
P05109
Homo sapiens


104
Glyceraldehyde-3-phosphate
G3P_HUMAN
36
kDa
P04406
Homo sapiens



dehydrogenase







105
Myosin-9
MYH9_HUMAN
227
kDa
P35579
Homo sapiens


106
Ig kappa chain V-III region VG
KV309_HUMAN
13
kDa
P04433
Homo sapiens



(Fragment)







107
Monocyte differentiation antigen
CD14_HUMAN
40
kDa
P08571
Homo sapiens



CD14







108
Heterogeneous nuclear
HNRH1_HUMAN
49
kDa
P31943
Homo sapiens



ribonucleoprotein H







109
ATP-dependent DNA helicase 2
KU70_HUMAN
70
kDa
P12956
Homo sapiens



subunit 1







110
Protein TFG
TFG_HUMAN
43
kDa
Q92734
Homo sapiens


111
Coagulation factor XII
FA12_HUMAN
68
kDa
P00748
Homo sapiens


112
Filaggrin-2
FILA2_HUMAN
248
kDa
Q5D862
Homo sapiens


113
Prothrombin
THRB_HUMAN
70
kDa
P00734
Homo sapiens


114
Sex hormone-binding globulin
SHBG_HUMAN
44
kDa
P04278
Homo sapiens


115
Heat shock cognate 71 kDa protein
HSP7C_HUMAN
71
kDa
P11142
Homo sapiens


116
Histidine-rich glycoprotein
HRG_HUMAN
60
kDa
P04196
Homo sapiens


117
Heterogeneous nuclear
ROA3_HUMAN
40
kDa
P51991
Homo sapiens



ribonucleoprotein A3







118
Ig kappa chain V-III region B6
KV301_HUMAN
12
kDa
P01619
Homo sapiens


119
Testis-expressed sequence 15
TEX15_HUMAN
315
kDa
Q9BXT5
Homo sapiens



protein







120
Polyadenylate-binding protein 1
PABP1_HUMAN
71
kDa
P11940
Homo sapiens


121
Lipopolysaccharide-binding protein
LBP_HUMAN
53
kDa
P18428
Homo sapiens


122
Lamina-associated polypeptide 2,
LAP2A_HUMAN
75
kDa
P42166
Homo sapiens



isoform alpha







123
Desmoplakin
DESP_HUMAN
332
kDa
P15924
Homo sapiens


124
Protein S100-A9
S10A9_HUMAN
13
kDa
P06702
Homo sapiens


125
Far upstream element-binding
FUBP2_HUMAN
73
kDa
Q92945
Homo sapiens



protein 2







126
Platelet glycoprotein lb alpha chain
GP1BA_HUMAN
69
kDa
P07359
Homo sapiens


127
Zinc finger and SCAN domain-
ZSC29_HUMAN
97
kDa
Q8IWY8
Homo sapiens



containing protein 29







128
Heterogeneous nuclear
HNRH3_HUMAN
37
kDa
P31942
Homo sapiens



ribonucleoprotein H3







129
Histone H3.3
H33_HUMAN
15
kDa
P84243
Homo sapiens


130
Stress-70 protein, mitochondrial
GRP75_HUMAN
74
kDa
P38646
Homo sapiens


131
Ig kappa chain V-I region AG
KV101_HUMAN
12
kDa
P01593
Homo sapiens


132
Elongation factor 1-alpha 1
EF1A1_HUMAN
50
kDa
P68104
Homo sapiens


133
RNA-binding protein FUS
FUS_HUMAN
53
kDa
P35637
Homo sapiens


134
DNA repair and recombination
RA54B_HUMAN
103
kDa
Q9Y620
Homo sapiens



protein RAD54B







135
Hornerin
HORN_HUMAN
282
kDa
Q86YZ3
Homo sapiens


136
Tubulin beta chain
TBB5_HUMAN
50
kDa
P07437
Homo sapiens


137
Heterogeneous nuclear
HNRPC_HUMAN
34
kDa
P07910
Homo sapiens



ribonucleoproteins C1/C2







138
RecQ-mediated genome instability
RMI1_HUMAN
70
kDa
Q9H9A7
Homo sapiens



protein 1







139
Desmocollin-1
DSC1_HUMAN
100
kDa
Q08554
Homo sapiens


140
Xaa-Pro dipeptidase
PEPD_HUMAN
55
kDa
P12955
Homo sapiens


141
Junction plakoglobin
PLAK_HUMAN
82
kDa
P14923
Homo sapiens


142
U3 small nucleolar RNA-associated
UT14C_HUMAN
87
kDa
Q5TAP6
Homo sapiens



protein 14 homolog C







143
Obscurin
OBSCN_HUMAN
868
kDa
Q5VST9
Homo sapiens


144
Elongation factor Tu, mitochondrial
EFTU_HUMAN
50
kDa
P49411
Homo sapiens


145
Heterogeneous nuclear
HNRPF_HUMAN
46
kDa
P52597
Homo sapiens



ribonucleoprotein F







146
Desmoglein-1
DSG1_HUMAN
114
kDa
Q02413
Homo sapiens


147
Heterogeneous nuclear
ROA1_HUMAN
39
kDa
P09651
Homo sapiens



ribonucleoprotein A1







148
ELAV-like protein 1
ELAV1_HUMAN
36
kDa
Q15717
Homo sapiens


149
Pyrroline-5-carboxylate reductase 1,
P5CR1_HUMAN
33
kDa
P32322
Homo sapiens



mitochondrial







150
Ig lambda chain V-I region WAH
LV106_HUMAN
12
kDa
P04208
Homo sapiens


151
Zinc finger and BTB domain-
ZBT10_HUMAN
92
kDa
Q96DT7
Homo sapiens



containing protein 10







152
Cystatin-C
CYTC_HUMAN
16
kDa
P01034
Homo sapiens


153
Major vault protein
MVP_HUMAN
99
kDa
Q14764
Homo sapiens


154
Phosphatidylcholine-sterol
LCAT_HUMAN
50
kDa
P04180
Homo sapiens



acyltransferase







155
Sulfhydryl oxidase 1
QSCN6_HUMAN
83
kDa
O00391
Homo sapiens


156
Far upstream element-binding
FUBP3_HUMAN
62
kDa
Q96124
Homo sapiens



protein 3







157
Ig delta chain C region
IGHD_HUMAN
42
kDa
P01880
Homo sapiens


158
Plasma serine protease inhibitor
IPSP_HUMAN
46
kDa
P05154
Homo sapiens


159
Heterogeneous nuclear
HNRPR_HUMAN
71
kDa
O43390
Homo sapiens



ribonucleoprotein R







160
Hemoglobin subunit gamma-1
HBG1_HUMAN
16
kDa
P69891
Homo sapiens




(+1)






161
TAR DNA-binding protein 43
TADBP_HUMAN
45
kDa
Q13148
Homo sapiens


162
Neural cell adhesion molecule Ll-
CHL1_HUMAN
135
kDa
O00533
Homo sapiens



like protein







163
Bifunctional polynucleotide
PNKP_HUMAN
57
kDa
Q96160
Homo sapiens



phosphatase/kinase







164
Fibrocystin
PKHD1_HUMAN
447
kDa
Q8TCZ9
Homo sapiens


165
Heterogeneous nuclear
ROAA_HUMAN
36
kDa
Q99729
Homo sapiens



ribonucleoprotein A/B







166
Peroxiredoxin-1
PRDX1_HUMAN
22
kDa
Q06830
Homo sapiens


167
40S ribosomal protein S16
RS16_HUMAN
16
kDa
P62249
Homo sapiens


168
Heat shock 70 kDa protein 1
HSP71_HUMAN
70
kDa
P08107
Homo sapiens


169
40S ribosomal protein S25
RS25_HUMAN
14
kDa
P62851
Homo sapiens


170
Fetuin-B
FETUB_HUMAN
42
kDa
Q9UGM5
Homo sapiens


171
Transferrin receptor protein 1
TFR1_HUMAN
85
kDa
P02786
Homo sapiens


172
Myelin basic protein
MBP_HUMAN
33
kDa
P02686
Homo sapiens


173
Aldo-keto reductase family 1
AK1C1_HUMAN
37
kDa
Q04828
Homo sapiens



member Cl







174
Uncharacterized protein C19orf21
CS021_HUMAN
75
kDa
Q8IV12
Homo sapiens


175
TATA-binding protein-associated
RBP56_HUMAN
62
kDa
Q92804
Homo sapiens



factor 2N







176
ATP-dependent DNA helicase 2
KU86_HUMAN
83
kDa
P13010
Homo sapiens



subunit 2







177
Sulfide:quinone oxidoreductase,
SQRD_HUMAN
50
kDa
Q9Y6N5
Homo sapiens



mitochondrial







178
Interleukin enhancer-binding factor
ILF2_HUMAN
43
kDa
Q12905
Homo sapiens



2







179
Prostaglandin F2-alpha receptor
PF2R_HUMAN
40
kDa
P43088
Homo sapiens


180
Peptidase inhibitor 16
PI16_HUMAN
49
kDa
Q6UXB8
Homo sapiens


181
Actin, alpha cardiac muscle 1
ACTC_HUMAN
42
kDa
P68032
Homo sapiens


182
E3 SUMO-protein ligase RanBP2
RBP2_HUMAN
358
kDa
P49792
Homo sapiens


183
Mitochondrial import inner
TIM50_HUMAN
40
kDa
Q3ZCQ8
Homo sapiens



membranetranslocase subunit








TIM50







184
Heterogeneous nuclear
HNRPD_HUMAN
38
kDa
Q14103
Homo sapiens



ribonucleoprotein DO







185
Interleukin enhancer-binding factor
ILF3_HUMAN
95
kDa
Q12906
Homo sapiens



3







186
Heterogeneous nuclear
HNRPG_HUMAN
42
kDa
P38159
Homo sapiens



ribonucleoprotein G







187
40S ribosomal protein S4, X isoform
RS4X_HUMAN
30
kDa
P62701
Homo sapiens


188
Caspase-14
CASPE_HUMAN
28
kDa
P31944
Homo sapiens


189
Annexin A2
ANXA2_HUMAN
39
kDa
P07355
Homo sapiens


190
Isocitrate dehydrogenase [NAD]
IDH3A_HUMAN
40
kDa
P50213
Homo sapiens



subunit alpha, mitochondrial







191
Cysteine-rich secretory protein 3
CRIS3_HUMAN
28
kDa
P54108
Homo sapiens


192
Myeloperoxidase
PERM_HUMAN
84
kDa
P05164
Homo sapiens


193
Transformation/transcription
TRRAP_HUMAN
438
kDa
Q9Y4A5
Homo sapiens



domain-associated protein







194
Isocitrate dehydrogenase [NAD]
IDH3B_HUMAN
42
kDa
O43837
Homo sapiens



subunit beta, mitochondrial







195
Collagen alpha-2(V) chain
CO5A2_HUMAN
145
kDa
P05997
Homo sapiens


196
Baculoviral IAP repeat-containing
BIRC6_HUMAN
528
kDa
Q9NR09
Homo sapiens



protein 6







197
Bleomycin hydrolase
BLMH_HUMAN
53
kDa
Q13867
Homo sapiens


198
Poly(rC)-binding protein 1
PCBP1_HUMAN
37
kDa
Q15365
Homo sapiens


199
Tubulin beta-3 chain
TBB3_HUMAN
50
kDa
Q13509
Homo sapiens


200
60 kDa heat shock protein,
CH60_HUMAN
61
kDa
P10809
Homo sapiens



mitochondrial







201
ATP synthase subunit beta,
ATPB_HUMAN
57
kDa
P06576
Homo sapiens



mitochondrial







202
40S ribosomal protein S18
RS18_HUMAN
18
kDa
P62269
Homo sapiens


203
DnaJ homolog subfamily A member
DNJA1_HUMAN
45
kDa
P31689
Homo sapiens



1







204
RNA-binding motif, single-stranded-
RBMS1_HUMAN
45
kDa
P29558
Homo sapiens



interacting protein 1







205
40S ribosomal protein S19
RS19_HUMAN
16
kDa
P39019
Homo sapiens


206
Tubulin alpha-1 chain
TBA1_HUMAN
50
kDa
P68366
Homo sapiens


207
Protein Hook homolog 1
HOOK1_HUMAN
85
kDa
Q9UJC3
Homo sapiens


208
Brother of CDO
BOC_HUMAN
121
kDa
Q9BWV1
Homo sapiens


209
Signal recognition particle 54 kDa
SRP54_HUMAN
56
kDa
P61011
Homo sapiens



protein







210
Trinucleotide repeat-containing 6B
TNC6B_HUMAN
194
kDa
Q9UP09
Homo sapiens



protein







211
Aldehyde dehydrogenase, dimeric
AL3A1_HUMAN
50
kDa
P30838
Homo sapiens



NADP-preferring







212
40S ribosomal protein S3
RS3_HUMAN
27
kDa
P23396
Homo sapiens


213
Fatty aldehyde dehydrogenase
AL3A2_HUMAN
55
kDa
P51648
Homo sapiens


214
Electron transfer flavoprotein
ETFA_HUMAN
35
kDa
P13804
Homo sapiens



subunit alpha, mitochondrial







215
RNA-binding protein EWS
EWS_HUMAN
68
kDa
Q01844
Homo sapiens


216
Histone H2A.V
H2AV_HUMAN
14
kDa
Q71UI9
Homo sapiens




(+1)






217
Heterogeneous nuclear
HNRPQ_HUMAN
70
kDa
O60506
Homo sapiens



ribonucleoprotein Q







218
Pyrroline-5-carboxylate reductase
P5CR2_HUMAN
34
kDa
Q96C36
Homo sapiens


219
Prohibitin
PHB_HUMAN
30
kDa
P35232
Homo sapiens


220
RuvB-like 1
RUVB1_HUMAN
50
kDa
Q9Y265
Homo sapiens


221
Protein-glutamine gamma-
TGM2_HUMAN
77
kDa
P21980
Homo sapiens



glutamyltransferase







222
Ubiquitin-associated protein 2
UBAP2_HUMAN
117
kDa
O5T6F2
Homo sapiens


223
Latent-transforming growth factor
LTB1L_HUMAN
187
kDa
Q14766
Homo sapiens



beta-binding protein 1







224
Thioredoxin domain-containing
TXND2_HUMAN
60
kDa
Q86VQ3
Homo sapiens



protein 2







225
Leucine-rich repeat-containing
LRC17_HUMAN
52
kDa
Q8N6Y2
Homo sapiens



protein 17







226
Protein FAM105B
F105B_HUMAN
40
kDa
Q96BN8
Homo sapiens


227
Myosin-la
MYO1A_HUMAN
118
kDa
Q9UBC5
Homo sapiens


228
Ras GTPase-activating-like protein
IQGA2_HUMAN
181
kDa
Q13576
Homo sapiens



IQGAP2







229
26S proteasome non-ATPase
PSMD1_HUMAN
102
kDa
Q99460
Homo sapiens



regulatory subunit 1







230
Actin, alpha skeletal muscle
ACTS_HUMAN
42
kDa
P68133
Homo sapiens


231
Cleavage stimulation factor subunit
CSTF3_HUMAN
83
kDa
Q12996
Homo sapiens



3







232
Clusterin
CLUS_HUMAN
52
kDa
P10909
Homo sapiens


233
Fibrocystin-L
PKHL1_HUMAN
466
kDa
Q86W11
Homo sapiens


234
InaD-like protein
INADL_HUMAN
196
kDa
Q8NI35
Homo sapiens


235
Keratin, type I cuticular Ha1
K1H1_HUMAN
47
kDa
Q15323
Homo sapiens


236
Keratin, type I cuticular Ha2
K1H2_HUMAN
50
kDa
Q14532
Homo sapiens


237
Keratin, type I cuticular Ha6
K1H6_HUMAN
52
kDa
O76013
Homo sapiens


238
Keratin, type I cytoskeletal 10
K1C10_HUMAN
59
kDa
P13645
Homo sapiens


239
Keratin, type I cytoskeletal 14
K1C14_HUMAN
52
kDa
P02533
Homo sapiens


240
Keratin, type I cytoskeletal 16
K1C16_HUMAN
51
kDa
P08779
Homo sapiens


241
Keratin, type I cytoskeletal 18
K1C18_HUMAN
48
kDa
P05783
Homo sapiens


242
Keratin, type I cytoskeletal 19
K1C19_HUMAN
44
kDa
P08727
Homo sapiens


243
Keratin, type I cytoskeletal 9
K1C9_HUMAN
62
kDa
P35527
Homo sapiens


244
Keratin, type II cuticular Hb1
KR181_HUMAN
55
kDa
Q14533
Homo sapiens


245
Keratin, type II cuticular Hb5
KR185_HUMAN
56
kDa
P78386
Homo sapiens


246
Keratin, type II cytoskeletal 1
K2C1_HUMAN
66
kDa
P04264
Homo sapiens


247
Keratin, type II cytoskeletal 1b
K2C1B_HUMAN
62
kDa
Q7Z794
Homo sapiens


248
Keratin, type II cytoskeletal 2
K22E_HUMAN
65
kDa
P35908
Homo sapiens



epidermal







249
Keratin, type II cytoskeletal 4
K2C4_HUMAN
57
kDa
P19013
Homo sapiens


250
Keratin, type II cytoskeletal 5
K2C5_HUMAN
62
kDa
P13647
Homo sapiens


251
Keratin, type II cytoskeletal 6B
K2C6B_HUMAN
60
kDa
P04259
Homo sapiens


252
Keratin, type II cytoskeletal 7
K2C7_HUMAN
51
kDa
P08729
Homo sapiens


253
Keratin, type II cytoskeletal 8
K2C8_HUMAN
54
kDa
P05787
Homo sapiens


254
Lymphoid-restricted membrane
LRMP_HUMAN
62
kDa
Q12912
Homo sapiens



protein







255
Myosin light chain 1, skeletal muscle
MLE1_HUMAN
21
kDa
P05976
Homo sapiens



isoform







256
Myosin-2
MYH2_HUMAN
223
kDa
Q9UKX2
Homo sapiens


257
Protein Shroom3
SHRM3_HUMAN
216
kDa
Q8TF72
Homo sapiens


258
Ras-related GTP-binding protein A
RRAGA_HUMAN
37
kDa
Q7L523
Homo sapiens


259
Sushi, nidogen and EGF-like
SNED1_HUMAN
152
kDa
Q8TER0
Homo sapiens



domain-containing protein 1







260
Retinol-binding protein 4
RET4_HUMAN
23
kDa
P02753
Homo sapiens
















TABLE 2







Proteins identified in spent media of human embryos.*
















Protein

Day of
Nature of


SEQID


Molecular
Accession
Embryo
Viability


NO.
Protein Name
Entry Name
Weight
Number
Development
Marker





261
14-3-3 protein zeta/delta
1433Z_HUMAN
 28 kDa
P63104
D1



262
6-phosphofructokinase, muscle
K6PF_HUMAN
 85 kDa
P08237
D1




type, isoform 1







263
6-phosphofructokinase, muscle
K6PF_HUMAN
 82 kDa
P08237-2
D1




type, isoform 1







264
Adenylate kinase isoenzyme 1
KAD1_HUMAN
 22 kDa
P00568
D1



265
Alpha-actinin-2
ACTN2_HUMAN
104 kDa
P35609
D1



266
Beta-enolase, isoform 1
ENOB_HUMAN
 47 kDa
P13929
D1



267
Beta-enolase, isoform 2
ENOB_HUMAN
 44 kDa
P13929-2
D1



268
Beta-enolase, isoform 3
ENOB_HUMAN
 42 kDa
P13929-3
D1



269
Creatine kinase M-type
KCRM_HUMAN
 43 kDa
P06732
D1



270
Fructose-bisphosphate aldolase A
ALDOA_HUMAN
 39 kDa
P04075
D1



271
Glycogen phosphorylase, muscle
PYGM_HUMAN
 97 kDa
P11217
D1




form







272
L-lactate dehydrogenase A chain
LDHA_HUMAN
 37 kDa
P00338
D1



273
Myosin-1
MYH1_HUMAN
223 kDa
P12882
D1



274
Myosin-2
MYH2_HUMAN
223 kDa
Q9UKX2
D1



275
Myosin-3
MYH3_HUMAN
224 kDa
P11055
D1



276
Myosin-4
MYH4_HUMAN
223 kDa
Q9Y623
D1



277
Myosin-6
MYH6_HUMAN
224 kDa
P13533
D1



278
Myosin-7
MYH7_HUMAN
223 kDa
P12883
D1



279
Myosin-8
MYH8_HUMAN
223 kDa
P13535
D1



280
Phosphoglucomutase-1, isoform 1
PGM1_HUMAN
 61 kDa
P36871
D1



281
Phosphoglucomutase-1, isoform 2
PGM1_HUMAN
 64 kDa
P36871-2
D1



282
Phosphoglycerate kinase 1
PGK1_HUMAN
 45 kDa
P00558
D1



283
Phosphoglycerate mutase 1
PGAM1_HUMAN
 29 kDa
P18669
D1



284
Pyruvate kinase isozyme M2
KPYM_HUMAN
 58 kDa
P14618
D1



285
Pyruvate kinase isozyme M1
KPYM_HUMAN
 58 kDa
P14618-2
D1



286
Sarcoplasmic/endoplasmic
AT2A1_HUMAN
110 kDa
O14983
D1




reticulum calcium ATPase 1,








Isoform SERCA1B







287
Sarcoplasmic/endoplasmic
AT2A1_HUMAN
 109 kDa
O14983-2
D1




reticulum calcium ATPase 1,








Isoform SERCA1A







288
Sarcoplasmic/endoplasmic
AT2A2_HUMAN
115 kDa
P16615
D1




reticulum calcium ATPase 2,








isoform 1







289
Sarcoplasmic/endoplasmic
AT2A2_HUMAN
110 kDa
P16615-2
D1




reticulum calcium ATPase 2,








isoform 2







290
Sarcoplasmic/endoplasmic
AT2A2_HUMAN
110 kDa
P16615-3
D1




reticulum calcium ATPase 2,








isoform 3







291
Sarcoplasmic/endoplasmic
AT2A2_HUMAN
112 kDa
P16615-4
D1




reticulum calcium ATPase 2,








isoform 4







292
Sarcoplasmic/endoplasmic
AT2A2_HUMAN
110 kDa
P16615-5
D1




reticulum calcium ATPase 2,








isoform 5







293
Triosephosphate isomerase, isoform
TPIS_HUMAN
 27 kDa
P60174
D1




1







294
Triosephosphate isomerase, isoform
TPIS_HUMAN
 27 kDa
P60174-21
D1




2







295
Tropomyosin alpha-1 chain, isoform
TPM1_HUMAN
 33 kDa
P09493
D1




1







296
Tropomyosin alpha-1 chain, isoform
TPM1_HUMAN
 27 kDa
P09493-2
D1




2







297
Tropomyosin alpha-1 chain, isoform
TPM1_HUMAN
 33 kDa
P09493-3
D1




3







298
Tropomyosin alpha-1 chain, isoform
TPM1_HUMAN
 33 kDa
P09493-4
D1




4







299
Tropomyosin alpha-1 chain, isoform
TPM1_HUMAN
 28 kDa
P09493-6
D1




5







300
Tropomyosin alpha-3 chain, isoform
TPM3_HUMAN
 33 kDa
P06753
D1




1







301
Tropomyosin alpha-3 chain, isoform
TPM3_HUMAN
 29 kDa
P06753-2
D1




2







302
Tropomyosin alpha-3 chain, isoform 
TPM3_HUMAN
 29 kDa
P06753-3
D1




3







303
Collagen alpha-1(I) chain
CO1A1_HUMAN
139 kDa
P02452
D1/D3



304
Cystatin-A
CYTA_HUMAN
 11 kDa
P01040
D1/D3



305
Filaggrin
FILA_HUMAN
435 kDa
P20930
D1/D3



306
Keratinocyte proline-rich protein
KPRP_HUMAN
 64 kDa
Q5T749
D1/D3



307
Selenium-binding protein 1, isoform
SBP1_HUMAN
 52 kDa
Q13228
D3




1







308
Selenium-binding protein 1, isoform
SBP1_HUMAN
 45 kDa
Q13228-2
D3




2







309
Fatty acid-binding protein, epidermal
FABP5_HUMAN
 15 kDa
Q01469
D1/D3/D5
Increased








with








implantation


310
Galectin-7
LEG7_HUMAN
 15 kDa
P47929
D1/D3/D5
Success








Increased








with








implantation


311
Ubiquitin
UBIQ_HUMAN
X kDa
P02248
D1/D3/D5
Success


312
Calmodulin-like protein 5
CALLS_HUMAN
 16 kDa
Q9NZT1
D1/D3/D5
Increased








with








implantation


313
14-3-3 protein sigma
1433S_HUMAN
 28 kDa
P31947
D1/D3/D5
Success


314
Histone H2A type 1-A
H2A1A_HUMAN
 14 kDa
Q96QV6
D1/D3/D5



315
Histone H3.1t
H311_HUMAN
 16 kDa
Q16695
D1/D3/D5



316
Histone H1.3
H13_HUMAN
 22 kDa
P16402
D3/D5
Increased








with








Aneuploidy


317
Lysozyme C
LYSC_HUMAN
 17 kDa
P61626
D3/D5
Increased








with








implantation


318
Plakophilin-1, isoform 2
PKP1_HUMAN
 83 kDa
Q13835
D3/D5
Success








Increased








with








implantation


319
Plakophilin-1, isoform 1
PKP1_HUMAN
 80 kDa
Q13835-2
D3/D5
Success


320
Abhydrolase domain-containing
AB12B_HUMAN
 41 kDa
Q7Z5M8
D3/D5




protein 12B, isoform 1







321
Abhydrolase domain-containing
AB12B_HUMAN
 32 kDa
Q7Z5M8-2
D3/D5




protein 12B, isoform 2







322
Abhydrolase domain-containing
AB12B_HUMAN
 29 kDa
Q7Z5M8-3
D3/D5




protein 12B, isoform 3







323
Abhydrolase domain-containing
AB12B_HUMAN
 13 kDa
Q7Z5M8-4
D3/D5




protein 12B, isoform 4







324
Abhydrolase domain-containing
AB12B_HUMAN
 25 kDa
Q7Z5M8-5
D3/D5




protein 12B, isoform 5







325
40S ribosomal protein SA
RSSA_HUMAN
 33 kDa
P08865
D5



326
Arginase-1, isoform 1
ARGI1_HUMAN
 35 kDa
P05089
D5



327
Arginase-1, isoform 2
ARGI1_HUMAN
 36 kDa
P05089-2
D5



328
Arginase-1, isoform 3
ARGI1_HUMAN
 25 kDa
P05089-3
D5



329
Beta-2-microglobulin
B2MG_HUMAN
 14 kDa
P61769
D5



330
Carbonic anhydrase 2
CAH2_HUMAN
 29 kDa
P00918
D5



331
Carboxypeptidase A4
CBPA4_HUMAN
 47 kDa
Q9UI42
D5



332
Catalase
CATA_HUMAN
 60 kDa
P04040
D5



333
Cathepsin D
CATD_HUMAN
 45 kDa
P07339
D5
Increased








with








implantation


334
CD44 antigen, isoform 1
CD44_HUMAN
 81 kDa
P16070
D5
Success








Increased








with








implantation


335
CD44 antigen, isoform 2
CD44_HUMAN
 3 kDa
P16070-2
D5
Success








Increased








with








implantation


336
CD44 antigen, isoform 3
CD44_HUMAN
 78 kDa
P16070-3
D5
Success








Increased








with








implantation


337
CD44 antigen, isoform 4
CD44_HUMAN
 77 kDa
P16070-4
D5
Success








Increased








with








implantation


338
CD44 antigen, isoform 5
CD44_HUMAN
 81 kDa
P16070-5
D5
Success








Increased








with








implantation


339
CD44 antigen, isoform 6
CD44_HUMAN
 77 kDa
P16070-6
D5
Success








Increased








with








implantation


340
CD44 antigen, isoform 7
CD44_HUMAN
 78 kDa
P16070-7
D5
Success








Increased








with








implantation


341
CD44 antigen, isoform 8
CD44_HUMAN
 74 kDa
P16070-8
D5
Success








Increased








with








implantation


342
CD44 antigen, isoform 9
CD44_HUMAN
 74 kDa
P16070-9
D5
Success








Increased








with








implantation


343
CD44 antigen, isoform 10
CD44_HUMAN
 53 kDa
P16070-10
D5
Success








Increased








with








implantation


344
CD44 antigen, isoform 11
CD44_HUMAN
 47 kDa
P16070-11
D5
Success








Increased








with








implantation


345
CD44 antigen, isoform 12
CD44_HUMAN
 39 kDa
P16070-12
D5
Success








Increased








with








implantation


346
CD44 antigen, isoform 13
CD44_HUMAN
 46 kDa
P16070-13
D5
Success








Increased








with








implantation


347
CD44 antigen, isoform 14
CD44_HUMAN
 43 kDa
P16070-14
D5
Success








Increased








with








implantation


348
CD44 antigen, isoform 15
CD44_HUMAN
 32 kDa
P16070-15
D5
Success








Increased








with








implantation


349
CD44 antigen, isoform 16
CD44_HUMAN
 73 kDa
P16070-16
D5
Success








Increased








with








implantation


350
CD44 antigen, isoform 17
CD44_HUMAN
 76 kDa
P16070-17
D5
Success


351
Creatine kinase B-type
KCRB_HUMAN
 43 kDa
P12277
D5



352
Dermokine, isoform 1
DMKN_HUMAN
 47 kDa
Q6E0U4
D5



353
Dermokine, isoform 2
DMKN_HUMAN
 47 kDa
Q6E0U4-2
D5



354
Dermokine, isoform 3
DMKN_HUMAN
 37 kDa
Q6E0U4-3
D5



355
Dermokine, isoform 4
DMKN_HUMAN
 45 kDa
Q6E0U4-4
D5



356
Dermokine, isoform 5
DMKN_HUMAN
 38 kDa
Q6E0U4-5
D5



357
Dermokine, isoform 6
DMKN_HUMAN
 42 kDa
Q6E0U4-6
D5



358
Dermokine, isoform 7
DMKN_HUMAN
 35 kDa
Q6E0U4-7
D5



359
Dermokine, isoform 8
DMKN_HUMAN
 20 kDa
Q6E0U4-8
D5



360
Dermokine, isoform 9
DMKN_HUMAN
 19 kDa
Q6E0U4-9
D5



361
Dermokine, isoform 10
DMKN_HUMAN
 17 kDa
Q6E0U4-
D5







10




362
Dermokine, isoform 11
DMKN_HUMAN
 15 kDa
Q6E0U4-
D5







11




363
Dermokine, isoform 12
DMKN_HUMAN
 16 kDa
Q6E0U4-
D5







12




364
Dermokine, isoform 13
DMKN_HUMAN
 14 kDa
Q6E0U4-
D5







13




365
Dermokine, isoform 14
DMKN_HUMAN
 15 kDa
Q6E0U4-
D5







14




366
Dermokine, isoform 15
DMKN_HUMAN
 10 kDa
Q6E0U4-
D5







15




367
Extracellular glycoprotein lacritin
LACRT_HUMAN
 14 kDa
Q9GZZ8
D5
Increased








with








implantation


368
Ferritin light chain (Ferritin L
FRIL_HUMAN
 20 kDa
P02792
D5
Success



subunit)







369
Fibrinogen gamma chain, isoform
FIBG_HUMAN
 52 kDa
P02679
D5




gamma-B







370
Fibrinogen gamma chain, isoform
FIBG_HUMAN
 49 kDa
P02679-2
D5
Increased



gamma-A




with








implantation


371
Gamma-enolase
ENOG_HUMAN
 47 kDa
P09104
D5
Success


372
Gamma-glutamylcyclotransferase,
GGCT_HUMAN
 21 kDa
O75223
D5




isoform 1







373
Gamma-glutamylcyclotransferase,
GGCT_HUMAN
 13 kDa
O75223-2
D5
Increased



isoform 2




with








implantation


374
Heat shock protein beta-1
HSPB1_HUMAN
 23 kDa
P04792
D5
Success


375
Histidine ammonia-lyase
HUTH_HUMAN
 73 kDa
P42357
D5



376
Ig mu chain C region, isoform 1
IGHM_HUMAN
 49 kDa
P01871
D5



377
Ig mu chain C region, isoform 2
IGHM_HUMAN
 52 kDa
P01871-2
D5



378
Interleukin-1 family member 7
IL1F7_HUMAN
224 kDa
Q9NZH6
D5




precursor, isoform B







379
Interleukin-1 family member 7
IL1F7_HUMAN
 22kDa
Q9NZH6-2
D5




precursor, isoform A







380
Interleukin-1 family member 7
IL1F7_HUMAN
 20 kDa
Q9NZH6-2
D5




precursor, isoform C







381
Interleukin-1 family member 7
IL1F7_HUMAN
 22 kDa
Q9NZH6-2
D5




precursor, isoform D







382
Interleukin-1 family member 7
IL1F7_HUMAN
 17 kDa
Q9NZH6-2
D5
Increased



precursor, isoform E




with








Aneuploidy


383
Lipocalin-1
LCN1_HUMAN
 19 kDa
P31025
D5



384
Methionine synthase
METH_HUMAN
141 kDa
099707
D5



385
Peroxiredoxin-2
PRDX2_HUMAN
 22 kDa
P32119
D5



386
Proprotein convertase
PCSK9_HUMAN
 74 kDa
Q8NBP7
D5




subtilisin/kexin type 9, isoform 1 







387
Proprotein convertase
PCSK9_HUMAN
 21 kDa
Q8NBP7-2
D5




subtilisin/kexin type 9, isoform 2 







388
Protein POF1B, isoform 1
POF1B_HUMAN
 69 kDa
Q8WVV4
D5



389
Protein POF1B, isoform 2
POF1B_HUMAN
 68 kDa
Q8WVV4-2
D5



390
Protein POF1B, isoform 3
POF1B_HUMAN
 35 kDa
Q8WVV4-3
D5
Increased








with








implantation


391
Protein S100-A7
S10A7_HUMAN
 11 kDa
P31151
D5
Success


392
Protein-glutamine gamma-
TGM3_HUMAN
 77 kDa
Q08188
D5




glutamyltransferase E







393
Pyruvate kinase isozymes M1/M2,
KPYM_HUMAN
 58 kDa
P14618
D5




isoform M2







394
Pyruvate kinase isozymes M1/M2,
KPYM_HUMAN
 58 kDa
P14618-2
D5




isoform M1







395
Receptor-type tyrosine-protein
PTPRG_HUMAN
162 kDa
P23470
D5




phosphatase gamma, isoform 1







396
Receptor-type tyrosine-protein
PTPRG_HUMAN
159 kDa
P23470-2
D5
Increased



phosphatase gamma, isoform 2




with








implantation


397
Serine protease inhibitor Kazal-type
ISK5_HUMAN
121 kDa
Q9NQ38
D5
Success



5, isoform f1




Increased








with








implantation


398
Serine protease inhibitor Kazal-type
ISK5_HUMAN
104 kDa
Q9NQ38
D5
Success



5, isoform short




Increased








with








implantation


399
Serine protease inhibitor Kazal-type
ISK5_HUMAN
124 kDa
Q9NQ38
D5
Success



5, isoform long







400
Serpin B3, isoform 1
SPB3_HUMAN
 45 kDa
P29508
D5



401
Serpin B3, isoform 2
SPB3_HUMAN
 39 kDa
P29508-2
D5
Increased








with








implantation


402
Uteroglobin
UTER_HUMAN
 10 kDa
P11684
D5
Success








Increased








with








implantation


403
V-set and immunoglobulin domain-
VSIG8_HUMAN
 44 kDa
Q5VU13
D5
Success



containing protein 8, isoform 1




Increased








with








implantation


404
V-set and immunoglobulin domain-
VSIG8_HUMAN
 25 kDa

D5
Success



containing protein 8, isoform 1





*All Proteins are from Homo Sapiens






Example 2
Secretome Profiling to Predict Implantation Potential of a Human Embryo
The following nonlimiting example teaches by way of illustration, not by limitation, the fabrication and employment of a customized immunoassay test kit for secretome profiling of a human embryo Immunoassay test kit fabrication occurs by modifying the well surfaces of a 96-well microtiter plate. Each separate well of the microtiter plate is incubated with an unlabelled capture antibody that recognizes one specific protein from the secretome panel. Custom capture antibodies are purchased from Rockland Immunochemicals, Inc. After incubation for 12 hours at 4° C., capture antibodies passively adsorb to the well surface. Subsequently, all wells of the 96-well plate are washed three times with a buffer comprising 1× phosphate buffer saline and 0.1% Tween-20 (PBST), blocked with 1% bovine serum albumin (BSA) for 1 hour at ambient temperature and washed three times with PBST.
Human cleavage-stage embryos are cultured in 10 μL drops of G1 supplemented with 2.5 mg/mL recombinant albumin under oil at 37° C., 6% CO2, 5% O2 for 24 hours. The embryos are washed twice in G2 culture media and further cultured in 10 μL drops of G2 supplemented with 2.5 mg/mL recombinant albumin under oil at 37° C., 6% CO2, 5% O2 for 48 hours with a fresh drop of G2 media added after 24 hours. Spent media samples of blastocysts are transferred into 0.65 mL Eppendorf tubes. Control groups comprise media cultured and collected under the same conditions but without embryos.
Secretome profiling proceeds using spent media culture in a sandwich ELISA format. The spent media culture is diluted into phosphate buffer saline (PBS) buffer and further distributed to separate wells of said customized microtiter plate. After incubation for one hour at room temperature, the samples are aspirated and all wells are washed four times with PBST buffer. Primary antibodies, each recognizing a distinct protein from the secretome panel, are distributed into wells that correspond to appropriate capture antibodies and permitted to incubate for one hour at room temperature. The wells are washed four times with PBST and incubated with a secondary antibody conjugated with an enzyme. The term “secondary antibody” refers to an antibody that binds to primary antibodies and may be conjugated with detection probes such as enzymes or fluorophores. In one embodiment, the secondary antibody contains the horseradish peroxidase (HRP) enzyme that converts the chromogenic substrate, 3,3′,5,5′-tetramethylbenzidene (TMB), into a blue product quantified at 655 nm on a spectrophotometer. In another embodiment, the HRP enzymatic reaction is stopped with a solution containing sulfuric acid and quantified at 450 nm.
In one embodiment, secretome profiling of a secretome panel of 261 individual proteins (Table 1), each selected for significant developmental competence and implantation potential, are monitored using said ELISA immunoassay test kit. In another embodiment, secretome profiling of a secretome panel comprising 37 proteins (Table 2) are monitored using said ELISA immunoassay test kit. Secretome profiling of the 37 individual proteins (Table 2) from spent media of human embryos correlates with embryonic viability and euploidy.
Changes may be made in the above methods and systems without departing from the scope hereof. It should be noted that the matter contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the present method and system and reasonable variations thereof, which, as a matter of language, might be said to fall therebetween.

## Claims

1. A method of predicting implantation success of a candidate embryo comprising:
contacting embryo secretions in a culture media with an immunosorbent assay kit that comprises a solid-state substrate functionalized with at least two distinct capture proteins, wherein the first capture protein specifically binds a protein having an amino acid sequence of SEQ ID NO: 310, and wherein the one or more additional capture proteins specifically bind a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-309 and 311-404, and
determining the secretome profile of a candidate embryo to ascertain the absence or presence of proteins implicated in implantation success;
wherein the presence of proteins implicated in implantation success predicts candidate embryo viability for implantation.

2. The method of claim 1, wherein the one or more additional capture proteins specifically bind a protein having an amino acid sequence selected from the group consisting of SEQ ID Nos. 261-309 and 311-404.

3. The method of claim 1, wherein the one or more additional capture proteins specifically bind a protein having an amino acid sequence selected from the group consisting of SEQ ID Nos. 311, 313, 317, 318, 319, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 383, 391, 397, 398, 399, 402, 403, and 404.

4. The method of claim 1, wherein the one or more additional capture proteins specifically bind a protein having an amino acid sequence selected from the group consisting of SEQ ID Nos. 311, 313, 318, 319, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 391, 397, 398, 399, 402, 403, and 404.

5. The method of claim 1, wherein the one or more additional capture proteins specifically bind a protein having an amino acid sequence selected from the group consisting of SEQ ID Nos. 317 and 383.