The neXt-50 Challenge

The neXt-50 Challenge

Chromosome 1

PIC Leaders: Ping Xu

Major lab members or partners contributing to the next-MP50: Ping Xu, Fuchu He, Yao Zhang, Feng Xu, Yanchang Li, Wei Wei, Yihao Wang, Xuehui Peng, Yang Chen, Shu Liu, Suzhen Li, Jinshuai Sun, Cuitong He, and Jiahui Shi

Status of the Chromosome “parts list” for your Chromosome:

According to the newest neXtProt (2018-01) dataset, we've updated the protein PE distribution of Ch1.

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision).

1) Jinshuai Sun, Jiahui Shi, Yihao Wang, Yang Chen, Yanchang Li, Degang Kong, Lei Chang, Fengsong Liu, Zhitang Lv, Yue Zhou, Fuchu He, Yao Zhang, Ping Xu. Multi-Proteases Combined with High-pH Reverse- Phase Separation Strategy Verified Fourteen Missing Proteins in Human Testis Tissue (pr-2018-00397q, revised) 2) Cuitong He, Jiahui Shi, Jinshuai Sun, Yihao Wang, Jialing Zhao, Shujia Wu, Yanchang Li, Lei Chang, Huiying Gao, Fengsong Liu, Zhitang Lv, Junzhu Wu, Fuchu He, Yao Zhang, Ping Xu. Digging for Missing Proteins Using Low-Molecular-Weight Protein Enrichment and a Mirror Protease Strategy (pr-2018-00398m, revised)

B) Titles and authors of papers published in the 2017 JPR SI.

1) Yihao Wang, Yang Chen, Yao Zhang, Wei Wei, Yanchang Li, Tao Zhang, Fuchu He, Yue Gao, Ping Xu. Multi-Protease Strategy Identifies Three PE2 Missing Proteins in Human Testis Tissue. 2017, 16(12): 4352- 4363. 2) Xuehui Peng, Feng Xu, Shu Liu, Suzhen Li, Qingbo Huang, Lei Chang, Lei Wang, Xin Ma, Fuchu He, Ping Xu. Identification of Missing Proteins in the Phosphoproteome of Kidney Cancer. 2017, 16(12): 4364-4373. C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years.

1) Cuitong He, Chenxi Jia, Yao Zhang, and Ping Xu. Enrichment-Based Proteogenomics Identifies Microproteins, Missing Proteins, and Novel smORFs in Saccharomyces cerevisiae. J. Proteome Res. 17, 7, 2335-2344

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers?

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According to the newest neXtProt (2018-01) dataset, nineteen MPs has been listed as PE1 proteins since HUPO-2017, 15 proteins were from Siqi Liu lab, three proteins from Ping Xu lab, and one protein from Gong Zhang lab.

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI.

According to the newest neXtProt (2018-01) dataset, three MPs has been listed as PE1 proteins since HUPO- 2017, two proteins (P0DMU9 and P0C5Z0) were from Wang, et al, and one protein (P81133) was from Peng, et al work.

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in).

In our pr-2018-00397q work, we also identified the other 39 MP candidates. It is worth to mention that we verified 4 MP candidates with one peptide containing more than 9 AA residues and another one containing only 8 AA residues, which is slightly lower than the criteria listed by C-HPP 2.1. In addition, we showed the high quality spectra of the 35 MP candidates with more than 1 unique peptides in supplementary figures (at least one peptide with more than 9 AA residues), which can be verified by peptide synthesis or other strategies. Among of these 39 MP candidates, 34 proteins were identified by at least two proteases digesting samples, and the other 5 proteins were identified by unique protease digestion.

In our pr-2018-00398m work, we also identified the other 4 MP candidates (Q0VDE8, Q2VIQ3, P0DMW4, Q8TBE1) with one unique peptide from LMW-enriching proteins, which showed high quality spectra and were verified by peptide synthesis. Among these 4 MP candidates, P0DMW4 was identified from the combination strategy of trypsin digestion and Gel-separating, and the other three proteins were from a single enriching strategy.

G) Any significant clinical or other successes are MP that you wish us to consider highlighting in the report.

The aim of pr-2018-00397q study was to dig more MPs by multi-protease method combined with high-pH RP separation based on large-scale proteomics. Surprisingly, we totally identified as many as 11,558 proteins from the pooled testis samples with three individuals, which is the largest proteome dataset for single human tissue sample so far, suggesting the huge effort we put on the C-HPP project this year. Taking advantage of this large dataset, we totally identified 100 MP candidates and verified 14 MPs. IPA analysis of these newly identified 14 MPs showed that 10 proteins were closely associated with liver disease and might be used for screening potential biomarkers for liver disease.

The aim of pr-2018-00398m study was to search small MP candidates based on LMW protein enriching strategy. To enrich these LMW proteins, we tried four different enriching strategies. To increase the identification credibility of LMW proteins, we utilized Trypsin and LysargiNase. Totally, we identified 4,063 proteins, which is a big proteome dataset specifically for the enriched LMW proteins. Among them, we identified seven MP candidates. The molecular weight of six MP candidates were lower than 40 kDa and four MP candidates were lower than 20 kDa. Importantly, these MP candidates with LWM can only be identified by the enriched LMW fractions but not in the largest testis proteome dataset, implying the efficiency of the enriching strategies applied in this study. This is the first time to introduce both of LMW proteins and mirror protease for testis sample in C- HPP, which may help to widespread use in the near future.

Chromosome 2

PIC Leaders: Lydie Lane

Major lab members or partners contributing to the next-MP50:

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Paula Duek (SIB/University of Geneva) Alain Gateau (SIB/University of Geneva) Thibault Robin (SIB/University of Geneva) Frédérique Lisacek (SIB/University of Geneva) Markus Mueller (SIB Lausanne) Amos Bairoch (SIB/University of Geneva) Charlotte Macron (Nestlé Institute of Health Sciences) Antonio Nunez-Galindo (Nestlé Institute of Health Sciences) Loïc Dayon (Nestlé Institute of Health Sciences)

Status of the Chromosome “parts list” for your Chromosome:

There were 130 MP on chr 2 in 2017, now only 98.

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision).

Related to the next-MP50 challenge:

Macron C, Lane L, Núñez Galindo A, Dayon L., Deep Dive on the Proteome of Human Cerebrospinal Fluid: A Valuable Data Resource for Biomarker Discovery and Missing Protein Identification. J Proteome Res. 2018 Aug 31. doi: 10.1021/acs.jproteome.8b00300. [Epub ahead of print] PMID: 30124047

Macron C, Lane L, Núñez Galindo A, Dayon L., Identification of Missing Proteins in Normal Human Cerebrospinal Fluid. J Proteome Res. 2018 Aug 17. doi: 10.1021/acs.jproteome.8b00194. [Epub ahead of print] PMID: 30106588

Robin T, Bairoch A, Müller M, Lisacek F, Lane L., Large-Scale Reanalysis of Publicly Available HeLa Cell Proteomics Data in the Context of the Human Proteome Project. J Proteome Res. 2018 Sep 17. doi: 10.1021/acs.jproteome.8b00392. [Epub ahead of print] PMID: 30175587

Related to the uPE1 challenge:

Duek P, Gateau A, Bairoch A, Lane L., Exploring the uncharacterized human proteome using neXtProt. J Proteome Res. 2018 Sep 7. doi: 10.1021/acs.jproteome.8b00537. [Epub ahead of print] PMID: 30191714

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Paik Y-K, Lane L, Kawamura T, Chen Y-J, Cho J-Y, LaBaer J, Yoo J S, Domont G, Corrales F, Omenn G, Arcjakov A, Encarnación-Guevara S, Liu S, Salekdeh G H, Cho J-Y, Kim C-Y, Overall C, Launching the C-HPP pilot project for functional characterization of identified proteins with no known function, in revision

Other papers related to HPP

Monti C, Lane L, Fasano M, Alberio T, An update of the functional mitochondrial human proteome network, J Proteome Res. 2018 Sep 19. doi: 10.1021/acs.jproteome.8b00447. [Epub ahead of print] PMID: 302303

Omenn GS, Lane L, Overall CM, Corrales FJ, Schwenk JM, Paik YK, Van Eyk JE, Liu S, Snyder M, Baker MS, Deutsch EW. Progress on Identifying and Characterizing the Human Proteome: 2018 Metrics from the HUPO Human Proteome Project. J Proteome Res. 2018 Aug 23. doi: 10.1021/acs.jproteome.8b00441. [Epub ahead of print] PMID: 30099871

B) Titles and authors of papers published in the 2017 JPR SI.

Carapito C, Duek P, Macron C, Seffals M, Rondel K, Delalande F, Lindskog C, Fréour T, Vandenbrouck Y, Lane L, Pineau C., Validating Missing Proteins in Human Sperm Cells by Targeted Mass-Spectrometry- and Antibody- based Methods. J Proteome Res. 2017 Dec 1;16(12):4340-4351. doi: 10.1021/acs.jproteome.7b00374. Epub 2017 Sep 26. PMID: 28891297

Omenn GS, Lane L, Lundberg EK, Overall CM, Deutsch EW., Progress on the HUPO Draft Human Proteome: 2017 Metrics of the Human Proteome Project. J Proteome Res. 2017 Dec 1;16(12):4281-4287. doi: 10.1021/acs.jproteome.7b00375. Epub 2017 Oct 9. PMID: 28853897

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years.

Schaeffer M, Gateau A, Teixeira D, Michel PA, Zahn-Zabal M, Lane L., The neXtProt peptide uniqueness checker: a tool for the proteomics community. Bioinformatics. 2017 Nov 1;33(21):3471-3472. doi: 10.1093/bioinformatics/btx318. PMID: 28520855

Gaudet P, Michel PA, Zahn-Zabal M, Britan A, Cusin I, Domagalski M, Duek PD, Gateau A, Gleizes A, Hinard V, Rech de Laval V, Lin J, Nikitin F, Schaeffer M, Teixeira D, Lane L, Bairoch A. The neXtProt knowledgebase on human proteins: 2017 update. Nucleic Acids Res. 2017 Jan 4;45(D1):D177-D182. doi: 10.1093/nar/gkw1062. Epub 2016 Nov 29.

Paik YK, Omenn GS, Hancock WS, Lane L, Overall CM. Advances in the Chromosome-Centric Human Proteome Project: looking to the future. Expert Rev Proteomics. 2017 Dec;14(12):1059-1071. doi: 10.1080/14789450.2017.1394189. Epub 2017 Nov 10. Review. PMID: 29039980

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers?

In Carapito et al. 2017, we reported the validation of 12 PE2 proteins by SRM and IHC. Due to an incomplete processing of SRM data in PeptideAtlas, only 4 of them were upgraded to PE1 in neXtProt in 2018.

In Robin et al. 2018, we reported the validation of 1 PE2 protein by reanalysing MS/MS data on 41 HeLa cell datasets.

In the two articles by Macron et al. 2018, we reported the validation of 14 PE2 proteins and 1 PE5 protein by analysing CSF by MS/MS

We expect that based on these 4 papers, 28 proteins will be upgraded to PE1 in Feb 2019.

In addition, the neXtProt team upgraded 6 proteins to PE1 based on high quality Ab-based studies, and 12 based on high quality mutagenesis studies.

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In total, our group has reported information allowing the validation of 46 proteins since the 2017 neXtProt reference release. 19 of them are on chromosome 2 (see table below).

Additionally, in Duek et al. 2018, we reported that 10 MPs actually correspond to non-coding elements (pseudogenes and lncRNAs) and should be deleted from the list of target MPs

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI.

22 MPs out of the 46 were upgraded in the 2018 neXtProt reference release (including 10 on chr 2). We expect the 24 others to be upgraded in the upcoming 2019 neXtProt reference release.

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in).

Too many to be listed here

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report.

Article Protein AC chr Status Feb 2017 Status Feb 2018 Carapito 2017 (targeted SRM/Ab-based study) A0AVI2 2 PE2 PE1

Q56UN5 2 PE2 PE1 Q8N7S2 2 PE2 PE2 Q8N6M5 2 PE2 PE2 A6NCI8 2 PE2 PE2 Q03828 2 PE2 PE2 Q9N2J8 2 PE2 PE2 O43506 14 PE2 PE1 Q6ZRR7 14 PE2 PE1 Q8N9Y4 14 PE2 PE2 A4IF30 14 PE2 PE2 C9J3V5 14 PE2 PE2 Robin 2018 (reanalysis of HeLa cell datasets from PRIDE) O75474 10 PE2 PE2 Macron 2018 (MS/MS analysis of CSF) Q9H1Z8 2 PE2 PE2

Q8NBL3 2 PE2 PE2 Q4G0M1 2 PE2 PE2 B5MCY1 2 PE2 PE2 Q9P2K9 1 PE2 PE2 Q9Y5E5 5 PE2 PE2 O60330 5 PE2 PE2 Q5BIV9 10 PE2 PE2 Q86VR8 11 PE2 PE2 Q8N6Y1 13 PE2 PE2 A6BM72 15 PE2 PE2 Q9ULX5 17 PE2 PE2 Q9NUR3 20 PE2 PE2 B8ZZ34 22 PE5 PE5

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Q8IUX8 X PE2 PE2 Manual curation, Ab based studies Q9HC97 2 PE2 PE1

Q8TAX0 2 PE2 PE1 Q92819 8 PE2 PE1 P0C7Q2 10 PE2 PE1 A5LHX3 14 PE2 PE1 Q6SJ96 14 PE2 PE1 Manual curation, mutagenesis studies Q9HB15 2 PE2 PE1

Q56UN5 2 PE2 PE1 P81277 2 PE2 PE1 P47872 2 PE2 PE1 Q9NZH7 2 PE2 PE1 Q9NS40 2 PE2 PE1 P81133 6 PE2 PE1 Q6V0L0 10 PE2 PE1 Q9HB14 14 PE2 PE1 Q9NPC1 14 PE2 PE1 Q8IYL9 14 PE2 PE1 K9M1U5 19 PE2 PE1

Chromosome 3 PIC Leaders: Takeshi Kawamura (Associate professor, The University of Tokyo)

Major lab members or partners contributing to the next-MP50: Lab member Kazuki Yamamoto (Assistant professor) Yoko Chikaoka (Researcher) SuJin Lee (Researcher)

Co-PI Toshihide Nishimur (Professor, St. Marianna University School of Medicine) Hiromasa Tojo (Professor, St. Marianna University School of Medicine)

Status of the Chromosome “parts list”:

■PE1 960 ■PE2 81 ■PE3 18 ■PE4 3 ■PE5 15

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision).

A simple method for selecting a representative protein with better protein evidence from a set of non-unique proteins in outputs of Percolator Fido. Hiromasa Tojo, Kiyonaga Fujii, Harubumi Kato, Toshihide Nishimura, Takeshi Kawamura

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(Submitted)

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in).

We have found four new proteins coded on chromosomes 1, 9, 17, and 20. However, only one peptide was detected, it doesn’t satisfy the requirement of the C-HPP Data Interpretation Guidelines. We have not found any for chromosome 3.

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report.

As a new approach, we are currently investigating that short mRNA products are translated into proteins. And the MP detection by irradiation of cells are also under investigation.

Chromosome 4 PIC Leaders: Yu-Ju Chen, Chia-Li Han, Ting-Yi Sung, Sung-Liang Yu

Major lab members or partners contributing to the next-MP50: Mehari Muuz Weldemariam, Reta Birhanu Kitata, Yu-Chang Tyan

Status of the Chromosome “parts list” for your Chromosome:

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision). “Subcellular Proteome Landscape of Human Embryonic Stem Cells Revealed Missing Membrane Proteins” Weldemariam MM, Han CL, Shekari F, Kitata RB, Chuang CY, Hsu WT, Kuo HC, Choong WK, Sung TY, He FC, Chung MCM, Salekdeh GH*, Chen YJ* (in press)

B) Titles and authors of papers published in the 2017 JPR SI. “Decoding the effect of isobaric substitutions on identifying missing proteins and variant peptides in human proteome” Choong WK, Lih TM, Chen YJ, Sung TY*

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. Not available

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers? 26 PE1-found MPs (gold MPs) in the dataset to be published in the 2018 special issue.

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI. In neXtProt 2017-01-23, the status for chromosome 4 missing proteins was 82. At the updated version, neXtProt

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2018-01-17, the number reduced to 71; suggesting 12 PE1-found MPs since 2017. In our report for hESC subproteomes in the JPR SI this year, we reported and verified 26 PE1-fuond (Gold) MPs which may be further promoted to PE1 proteins in the neXtProt.

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in). In the work reported in the coming SI, a total of 87 Silver MPs were identified in the hESC dataset. Among these proteins, 4 MPs are located in chromosome 4. The details are summarized in the following table. 1.

No Entry Gene name Protein Protein Chromosome #unique Peptide #RP #Subcellular # Evidence length No. peptides length fractions fractions PSMs 1 Q5BKT4 ALG10 PE2 473 12 1 10 5 3 44 2 Q6UXU6 TMEM92 PE2 159 17 1 13 4 2 30 3 A8MW99 MEI4 PE3 385 6 1 14 2 1 27 4 Q6ZUA9 MROH5 PE2 1318 8 1 18 3 3 18 5 Q9NUH8 TMEM14B PE2 114 6 1 29 4 2 18 6 Q9Y5E3 PCDHB6 PE2 794 5 1 12 1 1 17 7 Q9HBL6 LRTM1 PE2 345 3 1 9 1 1 14 8 Q9Y5H0 PCDHGA3 PE2 932 5 1 21 2 2 11 9 A1L157 TSPAN11 PE2 253 12 1 20 2 2 9 10 A6NLF2 ELOA3D PE3 546 18 1 12 2 1 9 11 Q2VPJ9 LRRC75B PE2 315 22 1 21 1 1 9 12 Q8WWZ4 ABCA10 PE2 1543 17 1 16 3 2 9 13 A6NFN9 ANKUB1 PE2 424 3 1 11 1 1 8 14 P05015 IFNA16 PE2 189 9 1 21 1 1 8 15 Q96AQ2 TMEM125 PE2 219 1 1 20 3 2 8 16 O95047 OR2A4 PE2 310 6 1 9 1 1 7 17 Q8NHC7 OR14C36 PE3 312 1 1 17 2 1 7 18 Q96CE8 TM4SF18 PE2 201 3 1 15 3 3 7 19 Q96LU7 MYRFL PE2 910 12 1 17 4 3 7 20 Q9NX77 ERVK13-1 PE2 482 16 1 14 1 1 7 21 A6NIZ1 NA PE2 184 5 1 27 1 1 6 22 A6NKW6 SHISAL2B PE3 160 5 1 13 2 1 6 23 B3SHH9 TMEM114 PE2 223 16 1 14 2 1 6 24 B7ZC32 KIF28P PE3 967 1 1 22 1 1 6 25 O75373 ZNF737 PE2 536 19 1 9 2 2 6 26 O75474 FRAT2 PE2 233 10 1 20 4 3 6 27 P0DJD1 RGPD2 PE2 1756 2 1 14 3 2 6 28 P46721 SLCO1A2 PE2 670 12 1 11 2 1 6 29 P60509 ERVPABLB- PE2 514 3 1 27 1 1 6 1 30 Q4KMZ8 NKAIN1 PE2 207 1 1 13 1 1 6 31 Q6QAJ8 TMEM220 PE2 160 17 1 12 3 3 6 32 Q8IV13 CCNJL PE2 435 5 1 11 1 1 6 33 Q8NH18 OR5J2 PE3 312 11 1 17 1 1 6 34 Q96T54 KCNK17 PE2 332 6 1 10 1 1 6 35 A8MUP2 CSKMT PE2 240 11 1 19 1 1 5 36 Q2VIQ3 KIF4B PE2 1234 5 1 36 1 1 5 37 Q92186 ST8SIA2 PE2 375 15 1 13 1 1 5 38 O76099 OR7C1 PE2 320 19 1 10 1 1 4 39 P78334 GABRE PE2 506 x 1 12 1 1 4 40 Q02045 MYL5 PE2 173 4 1 18 1 1 4 41 Q15546 MMD PE2 238 17 1 10 2 2 4 42 Q8IV35 WDR49 PE2 697 3 1 18 1 1 4 43 Q8IX29 FBXO16 PE2 292 8 1 11 1 1 4 44 Q8IYD2 KLHDC8A PE2 350 1 1 24 1 1 4 45 Q8N9F8 ZNF454 PE2 522 5 1 16 5 3 4 46 Q8NG84 OR2AK2 PE2 335 1 1 16 1 1 4 47 Q9H7M6 ZSWIM4 PE2 989 19 1 17 1 1 4 48 A6NNM3 RIMBP3B PE2 1639 22 1 34 1 1 3 49 A8MVX0 ARHGEF33 PE2 844 2 1 12 1 1 3 50 C9JSJ3 BHMG1 PE3 638 19 1 15 1 1 3 51 O75949 FAM155B PE2 473 x 1 10 1 1 3 52 P18825 ADRA2C PE2 462 4 2 29, 8 1 1 3 53 P60606 CTXN1 PE2 82 19 1 10 1 1 3 54 Q3B8N5 PROX2 PE2 592 14 1 12 1 1 3 55 Q495X7 TRIM60 PE2 471 4 1 12 1 1 3

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56 Q5GH73 XKR6 PE2 641 8 1 15 1 1 3 57 Q5T1S8 NCMAP PE2 102 1 1 35 1 1 3 58 Q5VVP1 SPATA31A6 PE3 1343 9 1 40 1 1 3 59 Q7Z444 ERAS PE2 233 x 1 23 1 1 3 60 Q86VR8 FJX1 PE2 437 11 1 20 1 1 3 61 Q86XJ0 CALHM3 PE2 350 10 1 18 1 1 3 62 Q8N1M1 BEST3 PE2 668 12 1 20 1 1 3 63 Q8NBL3 TMEM178A PE2 297 2 1 13 2 1 3 64 Q8WWF1 C1orf54 PE2 131 1 1 19 1 1 3 65 Q8WZ79 DNASE2B PE2 361 1 1 12 1 1 3 66 Q96KH6 C18orf12 PE2 178 18 1 16 1 1 3 67 Q96NR3 PTCHD1 PE2 888 x 1 13 1 1 3 68 Q9BX51 GGTLC1 PE2 225 20 1 38 1 1 3 69 Q9BZJ6 GPR63 PE2 419 6 1 10 1 1 3 70 Q9UN71 PCDHGB4 PE2 923 5 1 9 2 2 3 71 Q9Y5I1 PCDHA11 PE2 949 5 1 12 2 2 3 72 Q9Y5I3 PCDHA1 PE2 950 5 1 17 1 1 3 73 O43869 OR2T1 PE3 369 1 1 32 1 1 2 74 P0C264 SBK3 PE2 359 19 1 16 2 2 2 75 Q5T5M9 CCNJ PE2 372 10 1 10 1 1 2 76 Q5VXJ0 LIPK PE2 399 10 1 31 1 1 2 77 Q5W186 CST9 PE2 159 20 1 23 1 1 2 78 Q6B0B8 TIGD3 PE2 471 11 1 11 1 1 2 79 Q6ZTC4 NA PE2 211 20 1 14 1 1 2 80 Q8N7H1 LINC01465 PE2 131 12 1 28 1 1 2 81 Q8NG95 OR7G3 PE3 312 19 1 42 1 1 2 82 Q8NGU9 GPR150 PE3 434 5 1 33 1 1 2 83 Q8NH93 OR1L3 PE2 324 9 1 26 1 1 2 84 Q8TE23 TAS1R2 PE3 839 1 1 40 1 1 2 85 Q99742 NPAS1 PE2 590 19 1 10 2 1 2 86 Q9BPW5 RASL11B PE2 248 4 1 20 1 1 2 87 Q9P217 ZSWIM5 PE2 1185 1 1 24 1 1 2

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report.

Our deep subcellular proteomic profiles of hESC enabled us to map almost every molecule in the key pathways and network related to the regulation of hESCs. (1) Based on the mRNA expression data in HPA, the identified gold and silver MPs were involved in different lineage specific differentiations, suggesting their potential functional roles in developmental biology. Many developmental proteins were identified in each lineage. For example, three gold MPs, SLC10A3, TREM251, and TREM37, were involved in the lung development. (2) Many of them are involved in regulation of differentiation, self-renewal, epigenetic regulators, and cellular layers development through the core transcriptional networks in hESC. Interestingly, some identified MPs were associated with canonical pathways such as TGF-ß signaling (PCDHGA10-Gold MP, FRAT2-Silver MP) and MAPK signaling pathways (FRAT2-Silver MP, MMD-Silver MP), indicating their potential regulatory functions in hESC biology. Overexpression of FUT10 (Gold MP) has been reported to play a critical role in enhancement of self-renewal characteristics in embryonic stem cells [J. Biol. Chem. 2013, 288, 1076, 28859−28868]. In our data, this protein was confidently identified in more than one fractions (membrane and nucleus) and localized to Golgi membrane protein which was associated with STAT3 complex in LIF-AKT- STAT3 pathway. Thus, it may involve in regulating the core transcriptional networks by interacting with the key transcriptional factors including NANOG, SOX2 and OCT4 and eventually modulate self-renewal or cellular developments. G protein-coupled receptor 19 (GPR19, Gold MP), was reported among the list of 20 most positively significant genes in embryonic stem cells which may be crucial for maintaining or differentiating hESCs [Stem Cell Reports 2014, 10 (4), 472−479]. This Gold MP was identified in membrane fractions, consistent with its functional annotation as plasma protein receptor. Its role to regulate the GPCR signaling pathway and subsequently affect self-renewal or differentiation of hESCs remains further study.

Chromosome 5 PIC Leaders: Peter Horvatovich

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Major lab members or partners contributing to the next-MP50: Péter Horvatovich (bioinformatics, LC-MS analysis) Rainer Bischoff (clinical proteomics, PTM analysis)

Status of the Chromosome “parts list” for your Chromosome:

Proteins by chromosome & protein evidence

PE1: 775 (88.88%) PE2: 75 (8.60%) PE3: 9 (1.03%) PE4: 3 (0.34%) PE5: 10 (1.15%)

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision). None.

B) Titles and authors of papers published in the 2017 JPR SI. None.

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. • Klont, F., Bras, L., Wolters, J. C., Ongay, S., Bischoff, R., Halmos, G. B., Horvatovich, P. (2018). Assessment of sample preparation bias in mass spectrometry-based proteomics. Analytical Chemistry, 90(8), 5405-5413. DOI: 10.1021/acs.analchem.8b00600 [PMID 29608294]. • Güzel, C., Govorukhina, N., Wisman, G. B. A., Stingl, C., Dekker, L., Hollema, H., ... Luider, T. (2018). Proteomic alterations in early stage cervical cancer. Oncotarget, 9, 18128-18147. DOI: 10.18632/oncotarget.24773 [PMID 29719595] D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers? None. We did not checked this in particular, since the metric is done by the survival of Peptide Atlas identification at a very small FDR rate, which provide 1% FDR at protein level concerning the whole large dataset that PeptideAtlas is using in their human protein evidence annotations. Since the submitted papers were not submitted to JPR SI we do not have these numbers, but can be that our submitted dataset (mainly head and neck tissues) contributed to the improvement of human protein evidence status.

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI. None. See comment point E).

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in). None. See comment point E).

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report. Currently we are running number of proteogenomics and cancer related studies where we can contribute with SAAV, splice and many other structure variants of the human proteome. These studies (COPD, melanoma and head and neck cancer) are at various stages such as in preparation or submitted.

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Chromosome 6 PIC Leaders: Rob Moritz, Institute of Systems Biology, Seattle, WA (Cascadia Collaboration with UBC)

Major lab members or partners contributing to the neXt50: Eric Deutsch Ulrike Kusebauch Luis Mendoza David Shteynberg Michael Hoopmann

University of British Columbia, BC Canada Christopher Overall

Chromosome 7 PIC Leaders: Prof Ed Nice, Monash University

Major lab members or partners contributing to the neXt50: We are addressing the missing proteins, especially olfactory receptors using a BD approach in conjunction with MissingProteinPedia. Key labs involved are those of Prof Mark Baker (Macquarie) and Prof Ed Nice (Monash). This approach also has the potential to find missing proteins for other chromosomes.

Status of the Chromosome “parts list” for your Chromosome:

Relevant Papers. Accelerating the search for the missing proteins in the human proteome. Baker MS, Ahn SB, Mohamedali A, Islam MT, Cantor D, Verhaert PD, Fanayan S, Sharma S, Nice EC, Connor M, Ranganathan S. Nat Commun. 2017 Jan 24;8:14271. doi: 10.1038/ncomms14271.

Chromosome 8 PIC Leaders: Peng-yuan Yang

Major lab members or partners contributing to the next-MP50: Hua-li Shen, Xiao-hui Liu, Yang Zhang, Guo-quan Yan, Wei-min Zheng

Status of the Chromosome “parts list” for your Chromosome:

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A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision). None.

B) Titles and authors of papers published in the 2017 JPR SI. None.

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. A manuscript titled “Comparative analysis of human testes and epididymis through proteomic and RNA-seq methods” will be submitted soon. In this manuscript, we report 110 MPs. The authors are Wei-min Zheng, Xiao- hui Liu, Yang Zhang, Guo-quan Yan, Hua-li Shen and Peng-yuan Yang.

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers? In aforementioned paper, we have found 4 MPs since HUPO-2017 in Chromosome 8 (Q6PDA7, Q96LD1, Q9BXT5, Q9HAK2). Additionally, we have found 11MPs in Chromosome 1(A8MYJ7, O14904, P35325, P43115, Q5T089, Q5T1N1, Q5T9Z0, Q5TID7, Q7Z5Y6, Q8TDD5, Q86XG9) and 5 MPs in Chromosome 20 (Q8N688, Q9BQY6, Q9H106, Q9H4I0, Q6UDR6).

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI. For MPs in chromosome 8, 1 out of 4 is now in NeXt-Prot as PE1 proteins (Q9HAK2). For MPs in chromosome 1, 4 out of 11 are now in NeXt-Prot as PE1 proteins (O14904, P43115, Q5T089, Q8TDD5). For MPs in chromosome 20, all 5 are not PE1 proteins at the moment.

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in). We are working on it.

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report.

Not so far.

Chromosome 9 PIC Leaders: Je-Yoel Cho (Seoul Nat’l Univ.)

Major lab members or partners contributing to the neX-MPt50: Soo-Youn Lee (Samsung Medical Center) J. Eugene Lee (KRISS) Dong Wook Kim (Seoul Nat’l Univ.) Yong-In Kim (Seoul Nat’l Univ., KRISS) Hyoung-Min Park (Seoul Nat’l Univ.) Jisook Park (Samsung Medical Center)

Status of the Chromosome “parts list” for your Chromosome:

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670 (82.82%) 70 (8.65%)

21 (2.60%) 14 (1.73%) 34 (4.20%)

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision). "Launching the C-HPP pilot project for functional characterization of identified proteins with no known function“. YK Paik,…JY Cho…, C Overall.

B) Titles and authors of papers published in the 2017 JPR SI. None

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. Kim YI, and Cho JY. Gel-based proteomics in disease research: Is it still valuable? Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 2018

Park J, Lee GY, Choi JO, Jeon ES, Kim K, Kim JS, Lee SY. Development and Validation of Mass Spectrometry- Based Targeted Analysis for Amyloid Proteins. Proteomics Clin Appl. 2018

Park J, Lee E, Park KJ, Park HD, Kim JW, Woo HI, Lee KH, Lee KT, Lee JK, Park JO, Park YS, Heo JS, Choi SH, Choi DW, Jang KT, Lee SY. Large-scale clinical validation of biomarkers for pancreatic cancer using a mass spectrometry-based proteomics approach. Oncotarget. 2017

Aldonza MBD, Son YS, Sung HJ, Ahn JM, Choi YJ, Kim YI, Cho S, Cho JY. Paraoxonase-1 (PON1) induces metastatic potential and apoptosis escape via its antioxidative function in lung cancer cells. Oncotarget. 2017

Lee H, Chae S, Park J, Bae J, Go EB, Kim SJ, Kim H, Hwang D, Lee SW, Lee SY. Comprehensive Proteome Profiling of Platelet Identified a Protein Profile Predictive of Responses to An Antiplatelet Agent Sarpogrelate. Mol Cell Proteomics. 2016

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers? Not available

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI.

8 MPs at neXtProt 2017-08-01 are now annotated as PE1. One of them, C9orf66 (NX_Q5T8R8), is claimed by our previous JPR SI (Ahn JM et al., 2014).

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in). [Identified by MS2 scan – 5 MPs] C9orf50 (NX_Q5SZB4, Chr. 9) One unique peptide with length of 9 AAs. C9orf139 (NX_Q6ZV77, Chr. 9) One unique peptide with length of 12 AAs. RLN1 (NX_P04808, Chr. 9) One unique peptide with length of 13 AAs. DEFB123 (NX_Q8N688, Chr. 20) Two peptides were identified with length of 9 and 7 AAs. TEX15 (NXQ9BXT5, Chr. 8) One unique peptide with length of 12 AAs.

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[Detected by MRM – 16 MPs] ANKRD18A ( NX_Q8IVF6, Chr. 9) One unique peptide with length of 8 AAs ARIDC1-AS1(Chr. 9) One unique peptide with length of 11 AAs C9orf170 (NX_A2RU37, Chr. 9) One unique peptide with length of 11 AAs C9orf66 (NX_Q5T8R8, Chr. 9) One unique peptide with length of 9 AAs C9orf84 (NX_Q5VXU9, Chr. 9) One unique peptide with length of 13 AAs DEC1 (NX_Q9P2X7, Chr. 9) Two unique peptides with length of 10 AAs and 10 AAs FAM201A (NX_Q5SY85, Chr. 9) One unique peptide with length of 9 AAs FAM74A7 (NX_A6NL05, Chr. 9) One unique peptide with length of 10 AAs GLT6D1 (NX_Q7Z4J2, Chr. 9) One unique peptide with length of 13 AAs GVQW1 (NX_Q8N7I0, Chr. 9) One unique peptide with length of 14 AAs LCN8 (NX_Q6JVE9, Chr. 9) One unique peptide with length of 16 AAs LCN9 (NX_Q8WX39, Chr. 9) One unique peptide with length of 16 AAs SLC46A2 (NX_Q9BY10, Chr. 9) One unique peptide with length of 18 AAs SPATA31C1 (NX_P0DKV0, Chr. 9) One unique peptide with length of 9 AAs SPATA6L (NX_Q8N4H0, Chr. 9) Two unique peptides with length of 10 AAs and 12 AAs ZNF510 (NX_Q9Y2H8, Chr. 9) One unique peptide with length of 11 AAs

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report. Together with uPE1 functional annotation project (neXt-CP50), we tried to get MS evidence of MPs by IP-MS, which utilize overexpression in mammalian cell system. This approach could give us not only spectrum library for targeted MS, but interaction proteome information.

Chromosome 10 PIC Leaders: PI: Josh LaBaer Co-I: Jin Park

Major lab members or partners contributing to the next-MP50: Vel Murugan, Joe Micelie

Status of the Chromosome “parts list” for your Chromosome:

As of Sep, 2018

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision). NA

B) Titles and authors of papers published in the 2017 JPR SI. NA

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C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. NA

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers? 0

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI. 27

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in). 0

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report. As a member of the 5-chromosome consortium of Chr 5, 10, 15, 16, and 19, we have been providing the IVTT-compatible plasmids for missing proteins to other members for IVTT-assisted SRM and continue to generate more plasmids. We have assembled a comprehensive and one of the world’s largest collections of full- length plasmids representing more than 75% of all human protein-coding genes and are distributing the collection through our repository and distribution web portal DNASU (dnasu.org). Currently, we have full-length IVTT plasmids for around 50% of ~2,780 missing proteins and aim to cover >90% within the next year, which is available to the entire C-HPP team.

Chromosome 11 PIC Leaders: Dr. Jong Shin Yoo. (KBSI)

Major lab members or partners contributing to the next-MP50: Jin Young Kim (KBSI) Bong Hee Lee (Gacheon Univ.) Heeyoun Hwang (KBSI)

Status of the Chromosome “parts list” for your Chromosome:

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision). 1. Identification of Missing Proteins in Human Olfactory Epithelial Tissue by Liquid Chromatography- Tandem Mass Spectrometry, Heeyoun Hwang, Ji Eun Jeong, Hyun Kyoung Lee, Ki Na Yun, Hyun Joo An, Bonghee Lee, Young-Ki Paik, Tae Seok Jeong, Gi Taek Yee, Jin Young Kim, and Jong Shin Yoo, in press.

B) Titles and authors of papers published in the 2017 JPR SI.

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1. Next Generation Proteomic Pipeline for Chromosome-Based Proteomic Research Using NeXtProt and GENCODE Databases, Heeyoun Hwang, Gun Wook Park, Ji Yeong Park, Hyun Kyoung Lee, Ju Yeon Lee, Ji Eun Jeong, Sung-Kyu Robin Park, John R. Yates, Kyung-Hoon Kwon, Young Mok Park, Hyoung-Joo Lee, Young-Ki Paik, Jin Young Kim, and Jong Shin Yoo, 2017, 16, 4425-4434.

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. 1. Integrated Proteomic Pipeline Using Multiple Search Engines for a Proteogenomic Study with a Controlled Protein False Discovery Rate, Gun Wook Park, Heeyoun Hwang, Kwang Hoe Kim, Ju Yeon Lee, Hyun Kyoung Lee, Ji Young Park, Eun Sun Ji, Sung-Kyu Robin Park, John R. Yates, Kyung-Hoon Kwon, Young Mok Park, Hyoung-Joo Lee, Young-Ki Paik, Jin Young Kim, and Jong Shin Yoo, Journal of Proteome Research, 2016, 15, 4082-4090.

The PE level status of 15 Proteins in chromosome 11 were changed into PE1_2018 from PE2_2017. A list of them is shown as followed. Peptide Atlas Distinct Peptides DP in SRM Atlas DP in Protein Acc. and Name Status 2018-01b (DP) in Peptide References 2012-08 neXtProt_2018 human Atlas 2018-01b Edwards et al(2015) 1 Q9UL58 ZNF215 Canonical 32 10 33 + 2 Edwards et al(2015) 2 Q9UL59 ZNF214 Canonical 32 6 30 + 2 Hendriks et al 3 Q8IXT1 DDIAS Canonical 15 12 27 (2017)2 4 F8WCM5 INS-IGF2 Canonical 15 0 15 Omenn et al (2005) Kume et al (2014) + 5 Q96DL1 NXPE2 Subsumed by 4 6 9 4 Kume et al (2014) + 6 A6NK89 RASSF10 Canonical 7 5 9 11 Dammalli et al 7 P47874 OMP Canonical 6 6 8 (2017)1 Rieckmann et al 8 Q01362 MS4A2 Canonical 6 4 6 (2017)3 9 Q86XS5 ANGPTL5 not detected 0 5 5 N/A

10 Q96JQ5 MS4A4A Weak 1 1 2 Bell et al (2011)

11 Q6L8H4 KRTAP5-1 not detected 0 1 1 N/A indistinguishable 12 Q6L8G4 KRTAP5-11 1 0 1 Li et al (2017)4 representative 13 Q6L8H2 KRTAP5-3 not detected 0 1 1 N/A insufficient 14 Q6L8G8 KRTAP5-7 1 0 0 N/A evidence 15 O75690 KRTAP5-8 not detected 0 0 0 N/A

From these, 4 new report of missing proteins were published in other group.

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1. Proteomic Analysis of Human Olfactory Bulb, Manjunath Dammalli, Gourav Dey, Anil K. Madugundu, Manish Kumar, Benvil Rodrigues, Harsha Gowda, Bychapur Gowrishankar Siddaiah, Anita Mahadevan, Susarla Krishna Shankar, and Thottethodi Subrahmanya Keshava Prasad, OMICS, 2017, 21, 2. Site-Specific Mapping of the Human SUMO Proteome Reveals Co-Modification with Phosphorylation. Ivo A. Hendriks, David Lyon, Clifford Young, Lars J. Jensen, Alfred C. O. Vertegaal, and Michael L. Nielsen, Nature Structural and Molecular Biology, 2017, 24(3), 325-336 3. Social network architecture of human immune cells unveiled by quantitative proteomics, Jan C. Rieckmann, Roger Geiger, Daniel Hornburg, Tobias Wolf, Ksenya Kveler, David Jarrossay, Federica Sallusto, Shai S. Shen-Orr, Antonio Lanzavecchia, Matthias Mann and Felix Meissner, Nature Immunology, 2017, 18, 583-593 4. Digging More Missing Proteins Using an Enrichment Approach with ProteoMiner, Siqi Li, Yanbin He, Zhilong Lin, Shaohang Xu, Ruo Zhou, Feng Liang, Jian Wang, Huanming Yang, Siqi Liu, and Yan Ren, Journal of Proteome Research, 2017, 16(12), 4330-4339

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers? A total of 7 MPs was reported in our Chr. Group. Protein Acc. No. Peptides 2017-PE Now-PE Chr. Reporting Ref.

P0C7M7 NFNFAADVLDQWSQKEK PE2 PE2 12 Hwang et al. 2018

TGERPANPALWWVNGKGDEVK

HCLTGGEPLNPEVLEQWR

P46721 STVLKDDELKTKL PE2 PE2 12 Hwang et al. 2018

YGITKDFLPFMK

P59826 IDKDELGKAIQNSLVGEPILQNVLGSVTAVNR PE2 PE2 20 Hwang et al. 2018

AIQNSLVGEPILQNVLGSVTAVNR

GTPESLFELNSVMTVR

Q658L1 VTQNALFEGSTEFRESFQPWEIPPPEVK PE2 PE2 15 Hwang et al. 2018

SSVPFDDVTMYSVEYTPK

Q8N434 KLSLGTAEPQVKEPK PE2 PE2 7 Hwang et al. 2018

ALGMGTSGSLCR

Q16478 LYSAGAGGDAGSAHGGPQR PE2 PE1 19 Hwang et al. 2017

SFNYPSASLICAK

Q7LC44 QGEPLDQFLWR PE2 PE1 8 Hwang et al. 2017

EFLQYSEGTLSR

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI. 2 MPs of them were changed their status to PE1 level in now in neXtProt.

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in). There is 2 MP found, but not meeting the guideline 2.1. In our recent study (Hwang et al. 2018), 2 of FALSE POSITIVE Proteins of MP were identified using neXtProt DB only. But, due to using larger database in the nextPP approach including SAAV and GENCODE ASV, No false positives were identified. We showed tandem spectra of false positive MPs in Figure S1.

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G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report. The Guideline #12 of the validating analysis with synthetic peptides for extraordinary detection claims is very important.

For the breakthrough of finding MP 50, we need to analyse special human samples (e.g. Olfactory Epithelial Tissues) and develop an analytical method (e.g. membrane protein extracting method).

Chromosome 12 PIC Leaders: Ravi Sirdeshmukh. No report received.

Chromosome 13 PIC Leaders: Young-Ki Paik

Major lab members or partners contributing to the neXt50: Seul-Ki Jeong (Ph. D. , YPRC) Ju-Wan Kim (Ph.D. , YPRC) Jin-Young Cho (Ph. D. , YPRC) Keun Na (Ph. D., YPRC) Chae-Yeon Kim (Ph. D candidate)

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision). (1) “ASV-ID, a Proteogenomic Workflow to Predict the Candidate Protein Isoforms based on Transcript Evidence.” Jeong SK, Kim CY, Paik YK

B) Titles and authors of papers published in the 2017 JPR SI. (1) “Progress and Future Direction of Chromosome-Centric Human Proteome Project.” Paik YK, Overall CM, Deutsch EW, Van Eyk JE, Omenn GS (2) “Epsilon-Q: An Automated Analyzer Interface for Mass Spectral Library Search and Label-Free Protein

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Quantification.” Cho JY, Lee HJ, Jeong SK, Paik YK. (3) “Systematic Proteogenomic Approach To Exploring a Novel Function for NHERF1 in Human Reproductive Disorder: Lessons for Exploring Missing Proteins.” Na K, Shin H, Cho JY, Jung SH, Lim J, Lim JS, Kim EA, Kim HS, Kang AR, Kim JH, Shin JM, Jeong SK, Kim CY, Park JY, Chung HM, Omenn GS, Hancock WS, Paik YK

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. (1) “Advances in the Chromosome-Centric Human Proteome Project: looking to the future.” Paik YK, Omenn GS, Hancock WS, Lane L, Overall CM. Expert Rev Proteomics. 2017, 14(12):1059-1071

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers? 0 protein (see F entry below)

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI. 5 proteins are now PE1 but we did not claming them as candidate MPs because they were one-hit wonders in our studies. (see F entry below)

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in). 12 proteins are found as candidate MPs but not fulfilled the number of peptides

nextprot ac. guideline 2.1 PE (when publish) PE (2018-1-17) Comment

NX_Q5VVM6-1 Not satisfied, One-hit wonder 2 2 Cho JY et al., 2017

NX_Q8NGV6-1 Not satisfied, One-hit wonder 2 2 Cho JY et al., 2017

NX_Q8WXK1-1 Not satisfied, One-hit wonder 2 1 Cho JY et al., 2017

NX_Q8NE28-1 Not satisfied, One-hit wonder 2 1 Cho JY et al., 2017

NX_Q6IEU7-1 Not satisfied, One-hit wonder 2 2 Cho JY et al., 2017

NX_O75343-1 Not satisfied, One-hit wonder 2 5 Cho JY et al., 2017

NX_Q9H2F9-1 Not satisfied, One-hit wonder 2 1 Cho JY et al., 2017

NX_C9J6K1-1 Not satisfied, One-hit wonder 4 1 Cho JY et al., 2017

NX_Q96RP8-1 Not satisfied, One-hit wonder 2 2 Cho JY et al., 2017

NX_Q8N687-1 Not satisfied, One-hit wonder 2 2 Cho JY et al., 2017

NX_P57055-1 Not satisfied, One-hit wonder 2 1 Cho JY et al., 2017

NX_Q8N9B8 Not satisfied, One-hit wonder 2 2 Kim JW et al., 2016

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report. None

Chromosome 14 PIC Leaders: Charles Pineau Co-leader: Yves Vandenbrouck (BIG, BGE lab, CEA Grenoble)

Major lab members or partners contributing to the next-MP50: • Christine Carapito (LSMBO, University of Strabourg) • Charlotte Macron (Presently at: Nestlé Institute of Health Sciences) • Nathalie Melaine (Protim, Inserm, University of Rennes) • Emmanuelle Com (Protim, Inserm, University of Rennes) • Thomas Fréour (Reproductive Medicine unit, Nantes Hospital)

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• Major Partners : • Lydie Lane (SIB/University of Geneva) • Cecilia Lindskog-Bergström (Human Protein Atlas, Uppsala, Sweden)

Status of the Chromosome “parts list” for your Chromosome:

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision).

Related to the uPE1 challenge: Melaine N, Com E, Bellaud P, Guillot L, Lagarrigue M, Morrice NA, Guével B, Lavigne R, Velez de la Calle JF, Dojahn J, Pineau C. Deciphering the Dark Proteome: Use of the Testis and Characterization of Two Dark Proteins. J Proteome Res. 2018 Aug 31. doi: 10.1021/acs.jproteome.8b00387. PMID: 30130116

B) Titles and authors of papers published in the 2017 JPR SI.

Carapito C, Duek P, Macron C, Seffals M, Rondel K, Delalande F, Lindskog C, Fréour T, Vandenbrouck Y, Lane L, Pineau C., Validating Missing Proteins in Human Sperm Cells by Targeted Mass-Spectrometry- and Antibody- based Methods. J Proteome Res. 2017 Dec 1;16(12):4340-4351. doi: 10.1021/acs.jproteome.7b00374. Epub 2017 Sep 26. PMID: 28891297

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. N/A

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers?

In Carapito et al. 2017, we reported the validation of 12 PE2 proteins by LC-SRM and IHC. Due to an incomplete processing of SRM data in PeptideAtlas, only 4 of them were upgraded to PE1 in neXtProt in 2018 (2 on chr 2 and 2 on chr 14).

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI.

4 MPs out of the 12 were upgraded in the 2018 neXtProt reference release (including 2 on chr 14).

Article Protein AC chr Status Feb 2017 Status Feb 2018 Carapito et al., 2017 (Targeted SRM/Ab-based study) A0AVI2 2 PE2 PE1

Q56UN5 2 PE2 PE1 Q8N7S2 2 PE2 PE2 Q8N6M5 2 PE2 PE2 A6NCI8 2 PE2 PE2 Q03828 2 PE2 PE2 Q9N2J8 2 PE2 PE2 O43506 14 PE2 PE1 Q6ZRR7 14 PE2 PE1

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Q8N9Y4 14 PE2 PE2 A4IF30 14 PE2 PE2 C9J3V5 14 PE2 PE2

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in).

MPs identified and validated using LC-SRM or LC-PRM with the original peptide and (at least) one additional peptide (i.e., not observed using MS/MS). All these results have been reported and published in JPR HPP SI along with all experimental evidence.

Biological UniProt Reference MS-based validation PE Status PE Status in chr material AccNum method in neXtProt neXtProt Feb Feb 2017 2018 Human Sperm A2RUU4 Vandenbrouck et al., LC-PRM see Supplementary PE2 PE2 6 JPR 2016 Figure 1 Human Sperm A4IF30 Carapito et al., 2017 LC-PRM see Table 1 PE2 PE2 14 JPR Human Sperm A6NCI8 Carapito et al., 2017 LC-PRM see Table 1 PE2 PE2 2 JPR Human Sperm B2RC85 Vandenbrouck et al., LC-PRM see Supplementary PE2 PE2 7 JPR 2016 Figure 1 Human Sperm C9J3V5 Carapito et al., 2017 LC-PRM see Table 1 PE2 PE2 14 JPR Human Sperm Q03828 Carapito et al., 2017 LC-PRM see Table 1 PE2 PE2 2 JPR Human Sperm Q86X67 Vandenbrouck et al., LC-PRM see Supplementary PE2 PE2 10 JPR 2016 Figure 1 Human Sperm Q8N6M5 Carapito et al., 2017 LC-PRM see Table 1 PE2 PE2 2 JPR Human Sperm Q8N7S2 Carapito et al., 2017 LC-PRM see Table 1 PE2 PE2 2 JPR Human Sperm Q8N9Y4 Carapito et al., 2017 LC-PRM see Table 1 PE2 PE2 14 JPR Human Sperm Q96LI9 Vandenbrouck et al., LC-PRM see Supplementary PE2 PE2 X JPR 2016 Figure 1 Human Sperm Q9N2J8 Carapito et al., 2017 LC-PRM see Table 1 PE2 PE2 2 JPR Glioblastoma Q96HH4 Carapito et al., JPR LC-SRM see Figure 3 PE2 PE2 2 cells 2015

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report.

2 dark proteins characterized in the human testis (Melaine et al., 2018 JPR)

Chromosome 15 PIC Leaders: Gilberto Domont

Major lab members or partners contributing to the next-MP50:

Fabio Cesar Sousa Nogueira, Gustavo Monnerat, Erika Velasquez, Clarissa Ferolla, Natália Almeida

Status of the Chromosome “parts list” for your Chromosome:

PE1 506 86.36 % PE2 48 8.21 % PE3 14 2.23 % PE4 1 0.37 % PE5 31 2.84 %

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A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision). NONE

B) Titles and authors of papers published in the 2017 JPR SI.

Synaptosomal Proteome of the Orbitofrontal Cortex from Schizophrenia Patients Using Quantitative Label-Free and iTRAQ-Based Shotgun Proteomics VELÁSQUEZ, ERIKA ; Nogueira, Fabio C. S. ; VELÁSQUEZ, INGRID ; SCHMITT, ANDREA ; FALKAI, PETER ; Domont, Gilberto B. ; MARTINS-DE-SOUZA, DANIEL Journal of Proteome Research, v. 16, p. acs.jproteome.7b00422, 2017.

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. Proteomic assessment of colorectal cancers and respective resection margins from patients of the Amazon state of Brazil. ALMEIDA, FABIANA GREYCE OLIVEIRA; DE AQUINO, PRISCILA FERREIRA; CHALUB, SIDNEY RAIMUNDO S.; ARAUJO, GABRIEL DUARTE T.; Domont, Gilberto B.; DE SOUZA, AFONSO DUARTE L.; CARVALHO, PAULO C.; FISCHER, JULIANA DE SALDANHA DA G. Journal of Proteomics, v. 154, p. 59-68, 2017.

Quantitative proteomic analysis identifies proteins and pathways related to neuronal development in differentiated SH-SY5Y neuroblastoma cells JR Murillo, L Goto-Silva, A Sánchez, FCS Nogueira, GB Domont and Magno Junqueira EuPA Open Proteomics 16, 1-11

Application of iTRAQ Shotgun Proteomics for Measurement of Brain Proteins in Studies of Psychiatric Disorders EV Núñez, PC Guest, D Martins-de-Souza, GB Domont, FCS Nogueira Proteomic Methods in Neuropsychiatric Research, 219-227

Zika virus may not be alone: proteomics associates a bovine-like viral diarrhea virus to microcephaly FCS Nogueira, E Velasquez, ASO Melo, GB Domont bioRxiv, 062596

Mass spectrometry evaluation of a neuroblastoma SH-SY5Y cell culture protocol. Murillo JR, Pla I, Goto-Silva L, Nogueira FCS, Domont GB, Perez-Riverol Y, Sánchez A, Junqueira M. Anal Biochem. 2018 Oct 15; 559:51-54. doi: 10.1016/j.ab.2018.08.013. Epub 2018 Aug 23.

Synaptosomal Proteome of the Orbitofrontal Cortex from Schizophrenia Patients Using Quantitative Label-Free and iTRAQ-Based Shotgun Proteomics. Velásquez E, Nogueira FCS, Velásquez I, Schmitt A, Falkai P, Domont GB, Martins-de-Souza D. J Proteome Res. 2017 Dec 1;16(12):4481-4494. doi: 10.1021/acs.jproteome.7b00422. Epub 2017 Oct 13. iTRAQ-Based Shotgun Proteomics Approach for Relative Protein Quantification. Núñez EV, Domont GB, Nogueira FC. Methods Mol Biol. 2017; 1546:267-274.

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers? NONE

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI. NONE

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in). 2 candidates. All requested information is found in the attached spreadsheet

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G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report.

Protein unc-79 homologous isoform Iso 3 located on chromosome 14, initially with evidence only at transcript level, was identified with 2 unique peptides. The second protein, putative cytochrome b-c1 complex subunit Rieske-like protein 1 isoform Iso 1, belonging to chromosome 22, with evidence classified as uncertain, was identified with 1 unique peptide (Table S11 attached).

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Chromosome 16 PIC Leaders: Fernando J. Corrales

Major lab members or partners contributing to the next-MP50: Centro Nacional de Biotecnología, CSIC. Functional Proteomics laboratory. Fernando J. Corrales Universidad Complutense de Madrid. Proteomics Laboratory. Concha Gil. Centro de Investigación Médica Aplicada. Bioinformatics Laboratory. Víctor Segura

Status of the Chromosome “parts list” for your Chromosome:

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision).

Identification of the Missing Protein Hyaluronan Synthase 1 in Human Mesenchymal Stem Cells Derived from Adipose Tissue or Umbilical Cord. Clemente LF, Hernáez ML, Ramos-Fernández A, Ligero G, Gil C, Corrales FJ, Marcilla M. J Proteome Res. 2018 Jul 6. doi: 10.1021/acs.jproteome.8b00384. [Epub ahead of print]

Progress on Identifying and Characterizing the Human Proteome: 2018 Metrics from the HUPO Human Proteome Project. Omenn GS, Lane L, Overall CM, Corrales FJ, Schwenk JM, Paik YK, Van Eyk JE, Liu S, Snyder M, Baker MS, Deutsch EW. J Proteome Res. 2018 Aug 23. doi: 10.1021/acs.jproteome.8b00441. [Epub ahead of print]

B) Titles and authors of papers published in the 2017 JPR SI.

Enhanced Missing Proteins Detection in NCI60 Cell Lines Using an Integrative Search Engine Approach. Guruceaga E, Garin-Muga A, Prieto G, Bejarano B, Marcilla M, Marín-Vicente C, Perez-Riverol Y, Casal JI, Vizcaíno JA, Corrales FJ, Segura V. J Proteome Res. 2017 Dec 1;16(12):4374-4390. doi: 10.1021/acs.jproteome.7b00388. Epub 2017 Oct 11.

Prioritizing Popular Proteins in Liver Cancer: Remodelling One-Carbon Metabolism. Mora MI, Molina M, Odriozola L, Elortza F, Mato JM, Sitek B, Zhang P, He F, Latasa MU, Ávila MA, Corrales FJ. J Proteome Res. 2017 Dec 1;16(12):4506-4514. doi: 10.1021/acs.jproteome.7b00390. Epub 2017 Oct 2.

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. Progress and pitfalls in finding the 'missing proteins' from the human proteome map. Segura V, Garin-Muga A, Guruceaga E, Corrales FJ. Expert Rev Proteomics. 2017 Jan;14(1):9-14. doi: 10.1080/14789450.2017.1265450. Epub 2016 Dec 2. Review.

Liver cancer associated changes to the proteome: what deserves clinical focus? Mato JM, Elortza F, Lu SC, Brun V, Paradela A, Corrales FJ. Expert Rev Proteomics. 2018 Sep 11. doi: 10.1080/14789450.2018.1521277. [Epub ahead of print]

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The neXt-50 Challenge

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers? 1 (Hyaluronan synthase 1). It is encoded by a chr19 gene.

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI. Not yet annotated in neXtProt.

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in). 170 MP candidates. Among them, 2 were annotated as PE1 based on the results of the French-Swiss team before publication. For the other candidates, observations were based on a single peptide > 9 amino acids. No further validation has been done.

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report. We have published in the HPP JPR special issue several tools and strategies to facilitate the identification of the biological source (cell type or tissue) where the probability of finding MPs or a specific MP is optimal.

Chromosome 17 PIC Leaders: Gilbert S. Omenn, Michael P. Snyder

Major lab members or partners contributing to the next-MP50: Hongjiu Zhang, Omer Siddiqui, Chengxin Zhang, Yuanfang Guan

Status of the Chromosome “parts list” for your Chromosome: 44 new PE1 proteins since 2016 Taiwan Congress/Workshop (plus 6 candidates) 35 PE2,3,4 proteins prioritized for experimental detection (Siddiqui et al, JPR, 2018)

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision). Omenn GS, Lane L, Overall CM, Corrales FJ, Schwenk JM, Paik YK, Van Eyk JE, Liu S, Snyder M, Baker MS, Deutsch EW. Progress on Identifying and Characterizing the Human Proteome: 2018 Metrics from the HUPO Human Proteome Project. J Proteome Res. 2018 Aug 23. doi: 10.1021/acs.jproteome.8b00441. PMID: 30099871

Zhang C, Wei X, Omenn GS, Zhang Y. Structure anad protein interaction-based function annoations reveal likely functions of uncharacterized proteins in human chromosome 17. J Proteome Res (under final review).

Siddiqui O, Zhang H, Guan Y, Omenn GS. Chromosome 17 missing proteins: recent progress and future directions as part of the NeXt-50MP Challenge. J Proteome Res (under final review).

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Paik YK, Lane L, Kawamura T, Chen YJ, Cho JY, LaBaer J, Yoo SJ, Domont G, Corrales F, Omenn GS, Archakov A, Encarnacion-Guevara s, Liu S, Salekdeh GH, Cho JY, Kim CY, Overall CM. Launching the C-HPP pilot project for functional characterization of identified proteins with no known function. J Proteome Res (submitted).

B) Titles and authors of papers published in the 2017 JPR SI. Omenn GS, Lane L, Lundberg EK, Overall CM, Deutsch EW. Progress on the HUPO Draft Human Proteome: 2017 Metrics of the Human Proteome Project. J Proteome Res. 2017 Dec 1;16(12):4281-4287. doi: 10.1021/acs.jproteome.7b00375. PMID: 28853897

Paik YK, Overall CM, Deutsch EW, Van Eyk JE, Omenn GS. Progress and Future Direction of Chromosome- Centric Human Proteome Project. J Proteome Res. 2017 Dec 1;16(12):4253-4258. doi: 10.1021/acs.jproteome.7b00734. PMID: 29191025

Na K, Shin H, Cho JY, Jung SH, Lim J, Lim JS, Kim EA, Kim HS, Kang AR, Kim JH, Shin JM, Jeong SK, Kim CY, Park JY, Chung HM, Omenn GS, Hancock WS, Paik YK. Systematic Proteogenomic Approach To Exploring a Novel Function for NHERF1 in Human Reproductive Disorder: Lessons for Exploring Missing Proteins. J Proteome Res. 2017 Dec 1;16(12):4455-4467. doi: 10.1021/acs.jproteome.7b00146. PMID: 28960081

Schwenk JM, Omenn GS, Sun Z, Campbell DS, Baker MS, Overall CM, Aebersold R, Moritz RL, Deutsch EW. The Human Plasma Proteome Draft of 2017: Building on the Human Plasma PeptideAtlas from Mass Spectrometry and Complementary Assays. J Proteome Res. 2017 Dec 1;16(12):4299-4310. doi: 10.1021/acs.jproteome.7b00467. Epub 2017 Oct 10. PMID: 28938075

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. Zhou Y, Qin S, Sun M, Tang L, Yan X, Kilm TK, Caballero J, Glusman G, Brunkow ME, Soloski MJ, Rebman AW, Omenn GS, Moritz RL, Aucott JN, Hood L. Blood protein biomarkers that diagnose and classify idividuals with Lyme disease. (Submitted)

Li, H, Siddiqui O, Zhang H, Omenn GS, Guan Y. PIPA: Transfer learning improves the state of the art for protein abundance prediction. (submitted)

Zhang H, Zhu F, Dodge HH, Higgins GA, Omenn GS, Guan Y; Alzheimer's Disease Neuroimaging Initiative. A similarity-based approach to leverage multi-cohort medical data on the diagnosis and prognosis of Alzheimer's disease. Gigascience. 2018 Jul 1;7(7). doi: 10.1093/gigascience/giy085. PMID: 30010762

Guan Y, Li T, Zhang H, Zhu F, Omenn GS. Prioritizing Predictive Biomarkers for Gene Essentiality in Cancer Cells with mRNA Expression Data and DNA Copy Number Profile. Bioinformatics. 2018 Jun 15. doi: 10.1093/bioinformatics/bty467. PMID: 29912344

Li H, Panwar B, Omenn GS, Guan Y. Accurate prediction of personalized olfactory perception from large-scale chemoinformatic features. Gigascience. 2018 Feb 1;7(2). doi: 10.1093/gigascience/gix127. PMID: 29267859

Glusman G, Rose PW, Prlić A, Dougherty J, Duarte JM, Hoffman AS, Barton GJ, Bendixen E, Bergquist T, Bock C, Brunk E, Buljan M, Burley SK, Cai B, Carter H, Gao J, Godzik A, Heuer M, Hicks M, Hrabe T, Karchin R, Leman JK, Lane L, Masica DL, Mooney SD, Moult J, Omenn GS, Pearl F, Pejaver V, Reynolds SM, Rokem A, Schwede T, Song S, Tilgner H, Valasatava Y, Zhang Y, Deutsch EW. Mapping genetic variations to three- dimensional protein structures to enhance variant interpretation: a proposed framework. Genome Med. 2017 Dec 18;9(1):113. doi: 10.1186/s13073-017-0509-y. PMID: 29254494

Magis AT, Earls JC, Glusman G, Omenn GS, Lovejoy JC, Price ND, Hood L. Reply to "Precision medicine in the clouds". Nat Biotechnol. 2018 Aug 6;36(8):680-682. doi: 10.1038/nbt.4211. PMID: 30080845

Price ND, Magis AT, Earls JC, Glusman G, Levy R, Lausted C, McDonald DT, Kusebauch U, Moss CL, Zhou Y, Qin S, Moritz RL, Brogaard K, Omenn GS, Lovejoy JC, Hood L. A wellness study of 108 individuals using

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The neXt-50 Challenge personal, dense, dynamic data clouds. Nat Biotechnol. 2017 Aug;35(8):747-756. doi: 10.1038/nbt.3870. PMID: 28714965

Contreras-Galindo R, Fischer S, Saha AK, Lundy JD, Cervantes PW, Mourad M, Wang C, Qian B, Dai M, Meng F, Chinnaiyan A, Omenn GS, Kaplan MH, Markovitz DM. Rapid molecular assays to study human centromere genomics. Genome Res. 2017 Dec;27(12):2040-2049. doi: 10.1101/gr.219709.116. PMID: 29141960

Paik YK, Omenn GS, Hancock WS, Lane L, Overall CM. Advances in the Chromosome-Centric Human Proteome Project: looking to the future. Expert Rev Proteomics. 2017 Dec;14(12):1059-1071. doi: 10.1080/14789450.2017.1394189. PMID: 29039980

Rolland DCM, Basrur V, Jeon YK, McNeil-Schwalm C, Fermin D, Conlon KP, Zhou Y, Ng SY, Tsou CC, Brown NA, Thomas DG, Bailey NG, Omenn GS, Nesvizhskii AI, Root DE, Weinstock DM, Faryabi RB, Lim MS, Elenitoba-Johnson KSJ. Functional proteogenomics reveals biomarkers and therapeutic targets in lymphomas. Proc Natl Acad Sci U S A. 2017 Jun 20;114(25):6581-6586. doi: 10.1073/pnas.1701263114. PMID: 28607076

Seaman L, Chen H, Brown M, Wangsa D, Patterson G, Camps J, Omenn GS, Ried T, Rajapakse I. Nucleome Analysis Reveals Structure-Function Relationships for Colon Cancer. Mol Cancer Res. 2017 Jul;15(7):821-830. doi: 10.1158/1541-7786.MCR-16-0374. PMID: 28258094

Li H, Zhang Y, Guan Y, Menon R, Omenn GS. Annotation of Alternatively Spliced Proteins and Transcripts with Protein-Folding Algorithms and Isoform-Level Functional Networks. Methods Mol Biol. 2017;1558:415-436. doi: 10.1007/978-1-4939-6783-4_20. PMID: 28150250

Omenn GS. Advances of the HUPO Human Proteome Project with broad applications for life sciences research. Expert Rev Proteomics. 2017 Feb;14(2):109-111. doi: 0.1080/14789450.2017.1270763. PMID: 27935328

Omenn GS. The proteomes of the human eye, a highly compartmentalized organ. Proteomics. 2017 Jan;17(1- 2). doi: 10.1002/pmic.201600340. PMID: 27860232

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers? 43 from literature (see E) plus 1 from stranded peptide analysis (Siddiqui,et al, JPR 2018)

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI. 43 (see Siddiqui et al)

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in). 6 (stranded peptides found, but comparison of spectra with spectra in SRM Atlas not good enough)

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report.

(1) Analysis of progress on finding Chr 17 Missing Proteins (Siddiqui, Zhang, Guan, Omenn, JPR) The Chromosome-centric Human Proteome Project (C-HPP) announced in September 2016 an initiative to accelerate progress on detection and characterization of neXtProt PE2,3,4 “missing proteins” (MPs) with a mandate to each chromosome team to find about 50 MPs over two years. Here we report major progress toward the next-50 MP Challenge with 43 newly-validated Chr 17 PE1 proteins, of which 25 were based on mass spectrometry, 12 on protein-protein interactions, 3 on a combination of MS and PPI, and 3 with other types of data. Notable among these new PE1 proteins were 5 keratin-associated proteins, a single olfactory receptor, and five additional membrane-embedded proteins. We evaluate the prospects of finding the remaining 105 MPs coded for on Chr 17, focusing on mass spectrometry and protein-protein interaction approaches. We present a list of 35 prioritized MPs with specific approaches that may be used in further MS and PPI experimental studies. Additionally, we demonstrate how in silico studies can be used to capture individual peptides from major data

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The neXt-50 Challenge repositories, documenting 1MP that appears to be a strong candidate for PE1. We are close to our goal of finding 50 MPs for Chr 17.

(2) Predictions of functions for uPE1 proteins of Chr 17 Understanding the function of human proteins is essential to decipher the molecular mechanisms of human disease and phenotypes. Of the 17470 human protein coding genes in neXtProt 2018-01-17 database with unequivocal protein existences evidences (PE1), 1260 proteins do not have characterized functions. To reveal the function of poorly annotated human proteins, we developed a hybrid pipeline that creates protein structure prediction using I-TASSER and infers functional insights for the target protein from the functional templates recognized by COFACTOR. As a case study, the pipeline is applied to all 66 PE1 proteins with unknown or insufficiently specific function (uPE1) on human chromosome 17. Benchmark testing on a control set of 100 well-characterized proteins randomly selected from the same chromosome shows high Gene Ontology (GO) term prediction accuracies of 0.69, 0.57, and 0.67 for molecular function (MF), biological process (BP) and cellular component (CC), respectively. Three pipelines of function annotations (homology detection, protein- protein interaction network inference, and structure template identification) have been exploited by COFACTOR. Detailed analyses show that structure template detection based on low-resolution protein structure prediction made the major contribution to enhancement of the sensitivity and precision of the annotation predictions, especially for cases that do not have sequence-level homologous templates. For the Chr 17 uPE1 proteins, the I-TASSER/COFACTOR pipeline confidently assigned MF, BP and CC for 13, 33, and 49 proteins, respectively, with predicted functions ranging from sphingosine N-acyltransferase activity and sugar transmembrane transporter to cytoskeleton constitution. We highlight the 13 proteins with confident MF predictions; XX of these are among the 33 BP predictions. This study demonstrates a novel computational approach to systematically annotate protein function in the human proteome and provides useful insights to guide experimental design and follow-up validation studies of these uncharacterized proteins. We are prepared to extend this analysis to the entire proteome.

Chromosome 18 PIC Leaders: Alexander Archakov, Elena Ponomarenko (bioinformatics), Andrey Lisitsa (standardization)

Major lab members or partners contributing to the next-MP50: Victor G. Zgoda, Arthur T. Kopylov, Ekaterina V. Poverennaya, Ekaterina V. Ilgisonis, Stanislav N. Naryzhny Status of the Chromosome “parts list” for your Chromosome:

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision).

1) Increased Sensitivity of Mass-spectrometry by alkaline 2D-LC:It is Way to Deep Cover of Human Proteome in Gene-centric Mode. Ekaterina V. Ilgisonis, Arthur T. Kopylov, Elena A. Ponomarenko, Ekaterina V. Poverennaya, Olga V. Tikhonova, Tatiana E. Farafonova, Svetlana Novikova, Andrey V. Lisitsa, Victor G. Zgoda and Alexander I. Archakov – submitted to the JPR 2018 SI. Under revision 2) 200+ Protein Concentrations in Healthy Human Blood Plasma: Targeted Quantitative SRM SIS Screening of Chromosomes 18, 13, Y and the Mitochondrial Chromosome Encoded Proteome. Arthur T. Kopylov, Elena A. Ponomarenko, Ekaterina V. Ilgisonis, Mikhail A. Pyatnitskiy, Andrey V. Lisitsa, Ekaterina V. Poverennaya, Olga I. Kiseleva, Tatiana E. Farafonova, Olga V. Tikhonova, Maria G. Zavialova, Svetlana E. Novikova, Sergey A. Moshkovskii, Sergey P. Radko, Boris V. Morukov, Anatoly I. Grigoriev, Young-Ki Paik, Ghasem Hosseini Salekdeh, Andrea Urbani, Victor G. Zgoda and Alexander I. Archakov – submitted to the JPR 2018 SI. Under revision 3) Next Steps on In-Silico 2DE Analyses of Chr 18 Proteoforms. Naryzhny SN, Zorina E, Kopylov AT, Zgoda VG, Kleyst OA, Archakov AI. J Proteome Res. 2018 PubMed: 30238754 (Accepted).

B) Titles and authors of papers published in the 2017 JPR SI. 1) Poverennaya E.V., Ilgisonis E.V., Ponomarenko E.A., Kopylov A.T., Zgoda V.G., Radko S.P., Lisitsa A.V., Archakov A.I., Why Are the Correlations between mRNA and Protein Levels so Low among the 275 Predicted Protein-Coding Genes on Human Chromosome 18?, Journal of Proteome Research, 2017, 16(12), 4311-4318

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C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. 1) Poverennaya E.V., Kopylov A.T., Ponomarenko E.A., Ilgisonis E.V., Zgoda V.G., Tikhonova O.V., Novikova S.E., Farafonova T.E., Kiseleva Y.Y., Radko S.P., Vakhrushev I.V., Yarygin K.N., Moshkovskii S.A., Kiseleva O.I., Lisitsa A.V., Sokolov A.S., Mazur A.M., Prokhortchouck E.B., Skryabin K.G., Kostrjukova E.S., Tyakht A.V., Gorbachev A.Y., Ilina E.N., Govorun V.M., Archakov A.I., State of the Art on Chromosome 18- centric HPP in 2016: Transcriptome and Proteome Profiling of Liver Tissue and HepG2 Cells, Journal of Proteome Research, 2016, 15(11), 4030-4038

2) Kopylov A.T., Ilgisonis E.V., Moysa A.A., Tikhonova O.V., Zavialova M.G., Novikova S.E., Lisitsa A.V., Ponomarenko E.A., Moshkovskii S.A., Markin A.A., Grigoriev A.I., Zgoda V.G., Archakov A.I., Targeted Quantitative Screening of Chromosome 18 Encoded Proteome in Plasma Samples of Astronaut Candidates., Journal of Proteome Research, 2016, 15(11), 4039-4046

3) Naryzhny S.N., Maynskova M.A., Zgoda V.G., Ronzhina N.L., Kleyst O.A., Vakhrushev I.V., Archakov A.I., Virtual-experimental 2DE approach in chromosome-centric human proteome project, Journal of Proteome Research, 2016, 15(2), 525-530

4) Naryzhny S, Zgoda V, Kopylov A, Petrenko E, Kleist O, Archakov А. Variety and Dynamics of Proteoforms in the Human Proteome: Aspects of Markers for Hepatocellular Carcinoma. Proteomes. 2017 Nov 23;5(4). pii: E33. doi: 10.3390/proteomes5040033.

5) Naryzhny SN, Zgoda VG, Maynskova MA, Novikova SE, Ronzhina NL, Vakhrushev IV, Khryapova EV, Lisitsa AV, Tikhonova OV, Ponomarenko EA, Archakov AI. Combination of virtual and experimental 2DE together with ESI LC-MS/MS gives a clearer view about proteomes of human cells and plasma. Electrophoresis. 2016 Jan;37(2):302-9. doi: 10.1002/elps.201500382.

6) Ponomarenko EA, Poverennaya EV, Ilgisonis EV, Pyatnitskiy MA, Kopylov AT, Zgoda VG, Lisitsa AV, Archakov AI. The Size of the Human Proteome: The Width and Depth. Int J Anal Chem. 2016;2016:7436849. doi: 10.1155/2016/7436849.

7) Vialas V, Colomé-Calls N, Abian J, Aloria K, Alvarez-Llamas G, Antúnez O, Arizmendi JM, Azkargorta M, Barceló-Batllori S, Barderas MG, Blanco F, Casal JI, Casas V, de la Torre C, Chicano-Gálvez E, Elortza F, Espadas G, Estanyol JM, Fernandez-Irigoyen J, Fernandez-Puente P, Fidalgo MJ, Fuentes M, Gay M, Gil C, Hainard A, Hernaez ML, Ibarrola N, Kopylov AT, Lario A, Lopez JA, López-Lucendo M, Marcilla M, Marina- Ramírez A, Marko-Varga G, Martín L, Mora MI, Morato-López E, Muñoz J, Odena MA, de Oliveira E, Orera I, Ortea I, Pasquarello C, Ray KB, Rezeli M, Ruppen I, Sabidó E, Del Pino MMS, Sancho J, Santamaría E, Vazquez J, Vilaseca M, Vivanco F, Walters JJ, Zgoda VG, Corrales FJ, Canals F, Paradela A. A multicentric study to evaluate the use of relative retention times in targeted proteomics. J Proteomics. 2017 Jan 30;152:138- 149. doi: 10.1016/j.jprot.2016.10.014.

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers? No PE1-found MPs since HUPO-2017 were detected by chr18 team.

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI. Since HUPO-2017 (neXtProt 2017-08-01) 2 Mps were found at PE1.

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in). No candidate MPs were found either using the transcripto-proteomic approach (RNASeq and SRM) based on public data. The study of different types of biomaterial using alternative proteases Glu-C, Lys-C, AspN for improving of MP coverage is in the process now.

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report.

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The neXt-50 Challenge

In 2018 we aimed to measure the proteomic profiles and decipher the normal range of protein concentrations in the blood plasma of healthy people, including MPs. Unfortunately, no MPs were detected in the blood plasma of healthy people. During the next year the Chr 18 team's efforts will be focused on proteome profiling and MPs finding in the prostate cancer tissue and in obese human blood plasma.

To increase proteome coverage, a combination of shotgun technology and selected reaction monitoring with two-dimensional alkaline fractionation has been recently developed. To detect proteins that cannot be identified by such technologies, nanotechnologies such as combined atomic force microscopy with molecular fishing and/or nanowire detection may be useful.

Chromosome 19 PIC Leaders: Sergio Encarnación-Guevara

Contributing labs (Lab Heads named with affiliation): Ariadna Ortega, Leopolgo Gómez, Jeovanis Gil, Ramiro Alonso. Laboratory of Proteomics Center for Genomic Sciences UNAM. Julio Collado-Vides, Program of Computational Biology at Center for Genomic Sciences UNAM. Fernando Minauro Sanmiguel, Center for Medical Research in Human Genetics Alejandro García Carranca, National Cancer Institute Osbaldo Resendis Antonio, National Institute of Genomic Medicine. Guadalupe Ayala, National Institute of Public Health Partners: Fernando J. Corrales. ProteoRed, Spain.

Status of the Chromosome “parts list” for your Chromosome:

Since the C-HPP was launched in 2012, it has made substantial progress during the past 6 years. However, the Chromosome-centric Human Proteome Project (C-HPP) announced in September 2016 an initiative to accelerate progress on detection and characterization of neXtProt PE2,3,4 “missing proteins” (MPs) with a mandate to each chromosome team to find about 50 MPs over two years. In March 2017, we were invited to structure the Mexican consortium of chromosome 19, and began projects aimed at meeting the objectives of the global consortium in June 2017, for which we have 14 months working in fulfilling these objectives.

The chromosome 19 encodes for 1428, genes and most of them have been corroborated their existence at protein level. The neXtProt release 2016-01 listed 237 missing proteins on Chr 19. These missing proteins (MPs), are grouped in four categories according to their degree of confirmation. PE2 represents those genes that were confirmed at transcript level. PE3 correspond to genes inferred from homology. PE4 belong to the group of predicted and PE5 are classified as uncertain. The 237 MP of chromosome 19, 203 are in PE2, 29 in PE3, and 5 in PE4. More than 100 of CH19 MPs, are Zinc finger proteins, which show high homology among them, complicating the identification process. In addition, CH19, this is the second biggest human chromosome and the third containing more MPs.

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The neXt-50 Challenge

Certain families of genes are well represented on Chromosome 19, as mentioned two outstanding characteristics belong to this chromosome, more than 100 proteins MPs are Zinc finger proteins, which shown high homology among them, complicating the identification process. In addition, approximately the 10% of MPs of this chromosome are olfactory receptors.

For identification by MS, two non-overlapping proteotypic peptides of length at least 9 amino acids need to be observed and not be accounted for by sequence variants or isobaric PTMs of abundant PE1 proteins. The majority of MS is done with trypsin digestion; as such, it is important to note that, of the 2 MPs belonging to Chr 19, do not have two tryptic non-overlapping proteotypic peptides of length at least 9

Targeted Identification of MP using MS We performed an in-depth analysis in cervical cancer cell lines using different fractionation/separation protocols along with different protein extraction procedures with two aims, to contribute to the study stechiomety of lysine acetylation in cancer cells. And the other task and more important in this moment for us, the identification of the largest possible number of missing proteins belong to chromosome 19. In a first ongoing step, we decided to choose a strategy to characterize the lysine acetylation at proteome level. The method involves efficient total protein extraction and chemical acetylation of proteins with heavy isotopes, in order to differentiate them from endogenous acetylation. We also included Sodium Deoxycholate (SDC), a trypsin compatible detergent to avoid protein losses. We applied this strategy to the analysis of three cell lines treated with EX-527 (potent inhibitor of the lysine deacetylase SIRT1). In this study we identified more than 10,000 proteins including 22 MPs from CH19.

Three different cell lines were using in this analysis: HaCaT (non-cancerous immortalized human keratinocyte), SiHa (HPV-16 positive cervical cancer cell line) and CaLo (HPV-18 positive cervical cancer cell line).

All lysine residues of proteins were chemically acetylated with heavy isotopes, to differentiate them from endogenous acetylation. In consequence the lysine residues of all proteins are fully acetylated, and then the proteins were digested with trypsin. Due to trypsin cannot hydrolyze the peptide bond when acetyl-lysine is present, the generated peptides will be delimited by arginine residues, decreasing the de number of peptides in the sample. The mixtures of peptides were subjected to reverse phase chromatography, fractions were pooled together in 5 final fractions, LC-MS/MS Analysis, for MS analysis was performed in Dionex Ultimate 3000 RSLC nano UPLC system in-line coupled to a high resolution mass spectrometer Q-Exactive, the protein identification was performed using MaxQuant. So, Because we believe that MP is expressed in special or rare conditions we used 6-Chloro-2,3,4,9-tetrahydro-1 H-Carbazole-1-carboxamide (EX527) inhibitor of SIR1 (a potent inhibitor of the lysine deacetylase SIRT1, which has been linked to both tumor development, empowering a situation normally observed in cancer. Based in the strategy or approach mentioned before, a Bioinformatics analysis was carried out to characterize the MPs of the Chromosome 19 according to their feasibility to be identified by MS experiments. We performed an analysis considering those peptide with potential to be identified by mass spectrometry, based on their mass range, and using different strategies or enzymes appropriate to be used in large scale proteomics study. We compared the number of peptides and proteins that can be analyzed with normal trypsin digestion (hydrolyzing after Lysine and Arginine residues), the second was the strategy where at protein level all lysine residues are chemically acetylated. Trypsin is unable to hydrolyze the peptide bond after acetylated Lysine. In consequence the generated peptides with this strategy are delimited by Arginine residues; and the third uses Lys-C instead of trypsin, which hydrolyzes the peptide bond after Lysine residues. As expected the number of peptides generated with normal trypsin digestion is almost twice the number of peptides that can be analyze by the other strategies. However, the number of proteins is not significantly reduced.

With a reduction of more than 40% in the average of peptides per protein, we consider to be in better possibilities to identify less abundant proteins, as the case of MPs. Even when pre-fractionation steps are included, the number of peptides is overwhelming and those from most abundant proteins consume more time of the MS. The in silico analysis of the human proteome indicates that blocking lysine residues reduces the number peptide/protein to be analyzed from 32.8 to 19.5 without reducing the number of protein or MP that can be identified. In this sense a reduction in the number of peptides by restricting the cleavage sites of the enzyme will be highly valuable to our purpose of identifying MPs in Chromosome 19. Through MS/MS analysis, more than 10,000 distinct protein groups were identified, more than 9000 proteins for each cell line, you can see also on

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The neXt-50 Challenge the table below, the proteins expressed constant, up-regulated and down-regulated after treatment with SIRT1 inhibitor.

Mapping of proteins identified against the neXtProt database revealed 182 MPs; 149 PE2 with experimental evidence at the transcript level, 31 proteins inferred from homology(PE3), 2 protein predicted (PE4) totalizing 182, In addition we confirmed the expression of 22 MPs (PE2–PE4) belonging Chromosome 19, Figure 1.

Figure 1. Identified MPs distribution by chromosomes and biological material.

The previous study we have reviewed in more detail, since we consider the most fruitful and with strategies that we consider should be taken into account in subsequent studies that involve the identification of MPs.

To validate some missing proteins belong to the chromosome 19, 22 unique peptides were selected based on visual inspection of PSMs and synthesized. The peptides were mixed together and analyzed by LC–MS/MS (Q Exactive Plus, Thermo Fisher Scientific) to acquire higher energy collisional dissociation (HCD) fragmentation spectra for comparison with the initial spectra in the closest possible conditions. In the figure 2 we have a example of the our analyses to validate the missing proteins, we found a peptide from the MP zinc finger protein 135 endogenously acetylated in the K277 residue. Following this step, targeted assays using this approach were developed on the same LC–MS/MS platform to target 22 peptides belonging to 11 MPs, as we expected 10 of the 11 missing proteins validated using synthetic peptides are Zinc- finger proteins, and also 10 of 11 were detected expressed in cell treated with Sir1 inhibitor, EX527.

Ongoing projects. In Brian proteomics Fernando Minauro and his group is now working, specifically with gliobalstome cancer cell lines, mitochondria and vesicles produced by these cell lines in culture, this approach is being carried out, because more than 10 percent of missing proteins belonging to Chromosome 19 have been proposed as olfactory receptors, in consequence we believe the brains cells could be a good source of missing proteins. The experiments carried out demonstrate the identification of 9 MPs (2 in cell lines, 2 in mitochondria and 4 in vesicles) of which only 1 belongs to chromosome 19.

In response to C-HPP effort to detect and characterize the neXtProt PE2,3,4 “missing proteins”, we report big progress toward the next-50 MP Challenge with 289 MPs (PE2 216, PE3 70 and PE4 3) through the human genome, found during 18 months. Specifically, we show significant advances about chromosome 19 with 30 MPs (PE2 27 and PE3 3).

Ariadna Ortega and her group from National Institute of Genomic Medicine is participating in analyzing umbilical condom. MS/MS analysis of five umbilical cords, previously we separate artery, vein, and Wharton jelly, after protein extraction we pooled the proteins obtained from each condition and the MS/MS analysis was performed using three different protocols: (i) 1D SDS-PAGE separation (23 gel slices); (ii) protein digestion, and peptides analyzed by nano-LC with long gradient runs; and (iii) protein digestion, and peptides fractionated by high-pH reversed-phase (Hp-RP) chromatography. For all protocols, tryptic peptides were analyzed by high-resolution MS instruments (Q-Exactive). In this preliminary analysis we identified 60 MPs, artery 16, Wharton jelly 37 and vein, of which , belonging to Chromosome 19 we have (artery 1, Wharton jelly 2 and Vein 1). Figure 2 and Table 1.

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Guadalupe Ayala and colleagues, using iTRAQ approach combined with high-resolution mass spectrometry, analyzed biopsies from three stages: chronic gastritis, intestinal metaplasia, and gastric adenocarcinoma. They identified 3914 of which 22 were MPs and 2 of them identified as part of Chromosome 19 MPs , Figure 2 and Table 1.

Another approach was made by the group of Alejandro García Carranca using breast cancer cell lines identified 2 MPs of which 1 of them belongs to chromosome 19. Figure 2 and Table 1.

Chromosome 20 PIC Leaders: Siqi Liu

Major lab members or partners contributing to the next-MP50: Yan Ren, Zhilong Lin, Yanbin He, Siqi Li, Yuanliang Zhang.

Status of the Chromosome “parts list” for your Chromosome:

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision). Improvement of Peptide Separation for Exploring the Missing Proteins Localized on Membranes Yuanliang Zhang#, Zhilong Lin#, Siqi Liu*, Yan Ren* This manuscript is being revised.

B) Titles and authors of papers published in the 2017 JPR SI. Digging More Missing Proteins Using an Enrichment Approach with ProteoMiner. Siqi Li #, Yanbin He #, Zhilong Lin#, Siqi Liu*, Yan Ren*

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. NA

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers? 20 MPs were found and 15 MPs were verified by PRM through synthetic peptides in Digging More Missing Proteins Using an Enrichment Approach with ProteoMiner.

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI. All the 20 MPs found in our manuscript are listed as PE1 in NeXt-prot now.

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in). In our manuscript for 2018 C-HPP special issue, we announced 221 candidate MPs identified from human placenta and 2 human cancer cell lines, unfortunately in which only 30 of them (13.57%) passed the guideline 2.1. At the same time, 184(83.25%) and 7(3.17%) candidate MPs were excluded due to with only 1 unique peptide identified or with one or more peptides shorter than 9aa, respectively. All the peptide fractions in this project were injected into LC-MS for identification with duplicates. The identification reproducibility for the 184 and 7 MPs is 49.82% and 62% in two injections, respectively.

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report. NA

Chromosome 21 PIC Leaders: Albert Sickmann

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Major lab members or partners contributing to the next-MP50: No change

Status of the Chromosome “parts list” for your Chromosome: All: 252 PE1: 191 PE2: 22 PE3: 15 PE4: 1 PE5: 23

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision). No paper submitted.

B) Titles and authors of papers published in the 2017 JPR SI. No paper submitted

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. No paper submitted.

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers? 0

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI. 0

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in). 0

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report. 0

Status of the project: we have access to more than 30 samples from T21 patients. Before analysis an ethics approval is mandatory. We work on this since HuPO 2017.

Chromosome 22 PIC Leaders: Akhilesh Pandey No report.

Chromosome X PIC Leaders: PI: Yasushi Ishihama Co-PI: Tadashi Yamamoto

Major lab members or partners contributing to the neXt50: Amr Elguoshy Abdeldayem Shalaby (Niigata University) Shujiro Okuda (Niigata University) Shin Kawano (Database Center for Life Science, DBCLS) Susumu Goto (Database Center for Life Science, DBCLS) Masaki Matsumoto (Kyusyu University) Norie Araki (Kumamoto University)

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Status of the Chromosome “parts list” for your Chromosome:

PE1 : 706 (83.5%) PE2 : 87 (10.3%) PE3 : 18 (2.1%) PE4 : 6 (0.7%) PE5 : 29 (3.4%)

Total : 846

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (e.g. in press, or under revision). Update of Human Missing Protein Data Mining Strategy for “Stranded Peptides” in Public Proteome Databases Elguoshy A, Hirao Y, Xu B, Shimada S, Fujinaka H, Yamamoto K, Mitsui T, Yamamoto T (submitted)

B) Titles and authors of papers published in the 2017 JPR SI. Identification and Validation of Human Missing Proteins and Peptides in Public Proteome Databases: Data Mining Strategy. Elguoshy A, Hirao Y, Xu B, Saito S, Quadery AF, Yamamoto K, Mitsui T, Yamamoto T; Chromosome X Project Team of JProS. J Proteome Res. 2017 Dec 1;16(12):4403-4414. doi: 10.1021/acs.jproteome.7b00423. Epub 2017 Oct 31.

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. The jPOST environment: an integrated proteomics data repository and database. Moriya Y, Kawano S, Okuda S, Watanabe Y, Matsumoto M, Takami T, Kobayashi D, Yamanouchi Y, Araki N, Yoshizawa AC, Tabata T, Iwasaki M, Sugiyama N, Tanaka S, Goto S, Ishihama Y. Nucleic Acids Res. 2018 Oct 8. doi: 10.1093/nar/gky899. [Epub ahead of print]

The ProteomeXchange consortium in 2017: supporting the cultural change in proteomics public data deposition. Deutsch EW, Csordas A, Sun Z, Jarnuczak A, Perez-Riverol Y, Ternent T, Campbell DS, Bernal-Llinares M, Okuda S, Kawano S, Moritz RL, Carver JJ, Wang M, Ishihama Y, Bandeira N, Hermjakob H, Vizcaíno JA. Nucleic Acids Res. 2017 Jan 4;45(D1):D1100-D1106. doi: 10.1093/nar/gkw936. Epub 2016 Oct 18. jPOSTrepo: an international standard data repository for proteomes. Okuda S, Watanabe Y, Moriya Y, Kawano S, Yamamoto T, Matsumoto M, Takami T, Kobayashi D, Araki N, Yoshizawa AC, Tabata T, Sugiyama N, Goto S, Ishihama Y. Nucleic Acids Res. 2017 Jan 4;45(D1):D1107-D1111. doi: 10.1093/nar/gkw1080. Epub 2016 Nov 28.Proteome

Profiling of Diabetic Mellitus Patient Urine for Discovery of Biomarkers by Comprehensive MS-Based Proteomics. Hirao Y, Saito S, Fujinaka H, Miyazaki S, Xu B, Quadery AF, Elguoshy A, Yamamoto K, Yamamoto T. Proteomes. 2018 Feb 6;6(1). pii: E9. doi: 10.3390/proteomes6010009.

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers? Table 1. 41 Missing Proteins in the GPM validated with more than 2 “stranded peptides”

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E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI. 41 Missing Proteins in J Proteome Res. 2017 (16(12):4403-4414), which are validated with more than 2 peptides well-matched to those in the SRMAtlas (‘”stranded peptides”).

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in). 114 (155-41) other Missing proteins in J Proteome Res. 2017 (16(12):4403-4414), which are validated with 1 peptide well-matched to one in the SRMAtlas (‘”stranded peptides”) as shown in Table 2.

Table 2 Missing proteins validated with well-matched fragment profiles of more than 2 (blue lines) and 1 (white lines) peptides observed in GPM and SRMAtlas

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protein_ACC A B protein_ACC A B protein_ACC A B >Q86YR7_Gname=MCF2L2_PE=2 38 5 >Q5T953_Gname=IER5L_PE=2 8 1 >A8MXZ3_Gname=KRTAP9-1_PE=3 5 2 >Q96JF6_Gname=ZNF594_PE=2 27 3 >Q6ZMY9_Gname=ZNF517_PE=2 8 #N/A >O60224_Gname=SSX4;SSX4B_PE=2 5 #N/A >Q6ZN19_Gname=ZNF841_PE=2 20 3 >Q8N823_Gname=ZNF611_PE=2 8 1 >O75949_Gname=FAM155B_PE=2 5 1 >Q3ZCX4_Gname=ZNF568_PE=2 19 3 >Q99457_Gname=NAP1L3_PE=2 8 1 >Q13516_Gname=OLIG2_PE=2 5 1 >Q9Y6S9_Gname=RPS6KL1_PE=2 17 #N/A >Q9UK13_Gname=ZNF221_PE=2 8 #N/A >Q16534_Gname=HLF_PE=2 5 #N/A >Q86TJ5_Gname=ZNF554_PE=2 16 2 >Q9UL59_Gname=ZNF214_PE=2 8 #N/A >Q17RP2_Gname=TIGD6_PE=2 5 #N/A >P17039_Gname=ZNF30_PE=2 15 3 >O75290_Gname=ZNF780A_PE=2 7 1 >Q2VIQ3_Gname=KIF4B_PE=2 5 1 >Q8NBF1_Gname=GLIS1_PE=2 15 3 >P17021_Gname=ZNF17_PE=2 7 1 >Q6ICC9_Gname=LDOC1L_PE=2 5 #N/A >Q96BR6_Gname=ZNF669_PE=2 15 4 >Q13398_Gname=ZNF211_PE=2 7 1 >Q6TCH4_Gname=PAQR6_PE=2 5 1 >Q86TB3_Gname=ALPK2_PE=2 14 1 >Q15935_Gname=ZNF77_PE=2 7 #N/A >Q6ZNG0_Gname=ZNF620_PE=2 5 3 >Q96BV0_Gname=ZNF775_PE=2 14 3 >Q2M218_Gname=ZNF630_PE=2 7 #N/A >Q7Z3V5_Gname=ZNF571_PE=2 5 2 >P17025_Gname=ZNF182_PE=2 13 2 >Q52WX2_Gname=SBK1_PE=2 7 1 >Q86VR8_Gname=FJX1_PE=2 5 1 >P57058_Gname=HUNK_PE=2 13 1 >Q5T5D7_Gname=ZNF684_PE=2 7 1 >Q8N2I2_Gname=ZNF619_PE=2 5 1 >Q68CJ6_Gname=NUGGC_PE=2 13 1 >Q5U5X8_Gname=FAM222A_PE=2 7 1 >Q8N4W9_Gname=ZNF808_PE=2 5 2 >Q8NEK5_Gname=ZNF548_PE=2 13 1 >Q6P4F1_Gname=FUT10_PE=2 7 #N/A >Q8N7C4_Gname=TMEM217_PE=2 5 2 >Q8NFD2_Gname=ANKK1_PE=2 13 1 >Q6P9A1_Gname=ZNF530_PE=2 7 1 >Q8N972_Gname=ZNF709_PE=2 5 1 >Q96QS6_Gname=PSKH2_PE=2 13 3 >Q7LC44_Gname=ARC_PE=2 7 1 >Q8NEM1_Gname=ZNF680_PE=2 5 #N/A >Q86Y25_Gname=ZNF354C_PE=2 12 3 >Q7Z3E1_Gname=TIPARP_PE=2 7 2 >Q8WTX9_Gname=ZDHHC1_PE=2 5 #N/A >Q96SR6_Gname=ZNF382_PE=2 12 #N/A >Q86T29_Gname=ZNF605_PE=2 7 2 >Q8WUS8_Gname=SDR42E1_PE=2 5 #N/A >Q9UK10_Gname=ZNF225_PE=2 12 #N/A >Q8IVP9_Gname=ZNF547_PE=2 7 1 >Q9BYZ6_Gname=RHOBTB2_PE=2 5 #N/A >Q008S8_Gname=ECT2L_PE=2 11 1 >Q8N393_Gname=ZNF786_PE=2 7 #N/A >Q9H2H0_Gname=CXXC4_PE=2 5 1 >Q86XU0_Gname=ZNF677_PE=2 11 1 >Q8N6M9_Gname=ZFAND2A_PE=2 7 #N/A >Q9H7M6_Gname=ZSWIM4_PE=2 5 #N/A >Q92839_Gname=HAS1_PE=2 11 1 >Q8N859_Gname=ZNF713_PE=2 7 #N/A >Q9H7R5_Gname=ZNF665_PE=2 5 #N/A >Q96JL9_Gname=ZNF333_PE=2 11 2 >Q8TA94_Gname=ZNF563_PE=2 7 2 >Q9UKQ9_Gname=KLK9_PE=2 5 #N/A >Q96NG8_Gname=ZNF582_PE=2 11 1 >Q8TBH0_Gname=ARRDC2_PE=2 7 1 >Q9UL58_Gname=ZNF215_PE=2 5 #N/A >P17035_Gname=ZNF28_PE=2 10 1 >Q9BQ87_Gname=TBL1Y_PE=2 7 #N/A >Q9Y5G3_Gname=PCDHGB1_PE=2 5 #N/A >Q14588_Gname=ZNF234_PE=2 10 2 >Q9HCL3_Gname=ZFP14_PE=2 7 #N/A >A2A3L6_Gname=TTC24_PE=3 4 2 >Q8N7M2_Gname=ZNF283_PE=2 10 2 >Q9NP62_Gname=GCM1_PE=2 7 #N/A >A2RU54_Gname=HMX2_PE=2 4 #N/A >Q8TF20_Gname=ZNF721_PE=2 10 2 >Q9NS40_Gname=KCNH7_PE=2 7 #N/A >A5PLN7_Gname=FAM149A_PE=2 4 #N/A >Q96MI6_Gname=PPM1M_PE=2 10 2 >Q9UC06_Gname=ZNF70_PE=2 7 #N/A >A6NHJ4_Gname=ZNF860_PE=2 4 1 >Q96PE6_Gname=ZIM3_PE=2 10 3 >Q9UJN7_Gname=ZNF391_PE=2 7 #N/A >O76050_Gname=NEURL1_PE=2 4 2 >Q9UJU3_Gname=ZNF112_PE=2 10 #N/A >A6NJT0_Gname=UNCX_PE=3 6 1 >P15621_Gname=ZNF44_PE=2 4 #N/A >P52738_Gname=ZNF140_PE=2 9 #N/A >O75840_Gname=KLF7_PE=2 6 4 >Q06889_Gname=EGR3_PE=2 4 #N/A >P59817_Gname=ZNF280A_PE=2 9 3 >O76014_Gname=KRT37_PE=3 6 #N/A >Q14585_Gname=ZNF345_PE=2 4 1 >Q2VY69_Gname=ZNF284_PE=2 9 #N/A >Q03936_Gname=ZNF92_PE=2 6 1 >Q17RM4_Gname=CCDC142_PE=2 4 #N/A >Q3MIS6_Gname=ZNF528_PE=2 9 2 >Q6ZN06_Gname=ZNF813_PE=2 6 #N/A >Q3B7I2_Gname=CNPY1_PE=3 4 2 >Q6ZR37_Gname=PLEKHG7_PE=2 9 3 >Q6ZU67_Gname=BEND4_PE=2 6 #N/A >Q3KPI0_Gname=CEACAM21_PE=2 4 #N/A >Q6ZSS3_Gname=ZNF621_PE=2 9 4 >Q86UE3_Gname=ZNF546_PE=2 6 1 >Q49AA0_Gname=ZFP69_PE=2 4 1 >Q8IVC4_Gname=ZNF584_PE=2 9 3 >Q8IUN9_Gname=CLEC10A_PE=2 6 1 >Q5FWF6_Gname=ZNF789_PE=2 4 1 >Q8IXT1_Gname=DDIAS_PE=2 9 1 >Q8IUX8_Gname=EGFL6_PE=2 6 1 >Q5T089_Gname=MORN1_PE=2 4 #N/A >Q8N2E2_Gname=VWDE_PE=2 9 #N/A >Q8N9I5_Gname=FADS6_PE=2 6 #N/A >Q5T5M9_Gname=CCNJ_PE=2 4 1 >Q8NA42_Gname=ZNF383_PE=2 9 4 >Q8NDV1_Gname=ST6GALNAC3_PE=2 6 1 >Q5VIY5_Gname=ZNF468_PE=2 4 1 >Q9Y2G7_Gname=ZFP30_PE=2 9 #N/A >Q92819_Gname=HAS2_PE=2 6 2 >Q6UDR6_Gname=SPINT4_PE=3 4 #N/A >Q96HH4_Gname=TMEM169_PE=2 6 #N/A >Q6UWF9_Gname=FAM180A_PE=2 4 2 >Q96ND8_Gname=ZNF583_PE=2 6 #N/A >Q8IZF3_Gname=ADGRF4_PE=2 4 #N/A >Q9H1Z8_Gname=C2orf40_PE=2 6 #N/A >Q8N782_Gname=ZNF525_PE=2 4 #N/A >Q9NUC0_Gname=SERTAD4_PE=2 6 #N/A >Q8N8Z8_Gname=ZNF441_PE=2 4 #N/A >Q9NXS3_Gname=KLHL28_PE=2 6 #N/A >Q8NAM6_Gname=ZSCAN4_PE=2 4 #N/A >Q9UBC0_Gname=ONECUT1_PE=2 6 #N/A >Q8NDW4_Gname=ZNF248_PE=2 4 #N/A >Q9UQ07_Gname=MOK_PE=2 6 #N/A >Q8TAV4_Gname=STOML3_PE=2 4 1 >Q9Y5H3_Gname=PCDHGA10_PE=2 6 #N/A >Q8TB68_Gname=PRR7_PE=2 4 #N/A >Q9Y6R6_Gname=ZNF780B_PE=2 6 #N/A A: number of distinct peptides identified in GPM Db B: number of distinct peptides validated with SRMAtkas Db

Among 155 missing proteins identified in the GPM DB, 41 proteins (colored blue in the Table 1) are validated by more than 2 peptides, which fragment signatures are matched well to those found in the SRMAtlase DB (“Stranded peptides”). In a rank order of the number of distinct peptides, the 41 proteins have larger numbers of the peptides and some of the rest (114 proteins) also are identified with many distinct peptides.

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report. l We are still looking for Missing proteins in the GPM Db and the “Stranded peptides” by adapting a new more strict matching definition between fragmentation profiles from the GPM Db and SRMAtlas. l The jPOSTdb has now a series of tools for hunting missing proteins from new datasets deposited into jPOSTrepo, a member of ProteomeXchange Consortium under the jPOST environment, as follows: Ø jPOST missing protein search: 1. Visualize the distribution of PE1-5 categories automatically for each dataset with chromosome information. 2. Check the missing protein candidates using nextprot peptide uniqueness checker. Ø jPOST-iMPAQT db search for MRM transition setting 1. Check whether the MPs are within iMPAQT, MRM transition database. 2. Check mRNA expression of the MPs. 3. MRM assay for MP validation

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l PE2-4 contents (%) for each dataset in jPOSTdb can be used for the criteria to transfer to PeptideAtlas re- analysis pipeline.

Statistics of each dataset in jPOSTdb

n Number of detected proteins

n Protein evidence via neXtProt

Missing protein tools in jPOSTdb

n using latest & peptide uniqueness checker

Figure The jPOST workflow for hunting missing proteins

Chromosome Y PIC Leaders: Ghasem Hosseini Salekdeh

Major lab members or partners contributing to the next-MP50: Mehdi Mirzaei, Macquarie University, Australia Hossein Baharvand, Royan Institute, Iran

Status of the Chromosome “parts list” for your Chromosome:

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision).

1- Weldemariam MM, Han CL, Shekari F, Kitata RB, Chuang CY, Hsu WT, Kuo HC, Choong WK, Sung TY, He FC, Chung MCM, Salekdeh GH, Chen YJ. Subcellular Proteome Landscape of Human Embryonic Stem Cells Revealed Missing Membrane Proteins. J Proteome Res. 2018 Sep 11. doi: 10.1021/acs.jproteome.8b00407.

2- Kopylov, Arthur; Ponomarenko E, Ilgisonis E, Pyatnitskiy M, Lisitsa A, Poverennaya E, Kiseleva O, Farafonova T, Tikhonova O, Zavialova M, Novikova S, Moshkovskii S, Radko S, Morukov B, Grigoriev A, Paik YK, Salekdeh GH, ; Urbani A, Zgoda V, Archakov A. 200+ Protein Concentrations in Healthy Human

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Blood Plasma: Targeted Quantitative SRM SIS Screening of Chromosomes 18, 13, Y and the Mitochondrial Chromosome Encoded Proteome. J Proteome Res. (Revised manuscript submitted)

B) Titles and authors of papers published in the 2017 JPR SI.

1- Meyfour A, Pooyan P, Pahlavan S, Rezaei-Tavirani M, Gourabi H, Baharvand H, Salekdeh GH. Chromosome- Centric Human Proteome Project Allies with Developmental Biology: A Case Study of the Role of Y Chromosome Genes in Organ Development. J Proteome Res. 2017 Dec 1;16(12):4259-4272.

2- Meyfour A, Ansari H, Pahlavan S, Mirshahvaladi S, Rezaei-Tavirani M, Gourabi H, Baharvand H, Salekdeh GH. Y Chromosome Missing Protein, TBL1Y, May Play an Important Role in Cardiac Differentiation. J Proteome Res. 2017 Dec 1;16(12):4391-4402.

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years.

Meyfour A, Pooyan P, Pahlavan S, Rezaei-Tavirani M, Gourabi H, Baharvand H, Salekdeh GH. Chromosome- Centric Human Proteome Project Allies with Developmental Biology: A Case Study of the Role of Y Chromosome Genes in Organ Development. J Proteome Res. 2017 Dec 1;16(12):4259-4272.

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers?

None in Chr Y but we have found MPs of other chromosomes:

1) In sperm samples (unpublished data) we could identified Testis-expressed protein 49 (TBX9) (PE4 of Chromosome 12) with more than two peptides with aa ≥ 9

2) In collaboration with YuJu Chen, we identified 26 MPs with more than two peptides with aa ≥ 9 in subcellular fractions of human embryonic stem cell (hESCs)(Weldemariam et al., JPR SI, 2018)). These include MPs of Chr 1 (4 MPs), Chr 2 ( 1 MP), Chr 3 (2 MPs), Chr 4 (1 MP), Chr 5 (1 MP), Chr 6 (1 MP), Chr 8 (1 MP), Chr 9 (1 MP), Chr 12 (1 MP), Chr 13 (1 MP), Chr 14 (1 MP), Chr 15 (1 MP), Chr 19 (5 MP), Chr 22 (1 MP) and Chr X (4 MP).

Table 1- 26 MPs identified in subcellular fraction of hESCs (Weldemariam et al., JPR SI, 2018) No. Entry Gene Name Protein Protein Chromosome #Unique Peptides Evidence Length No. 1 Q6TDP4 KLHL17 PE2 642 1 2 2 Q9P2K9 DISP3 PE2 1392 1 2 3 Q8WXS4 TMEM37 PE2 190 1 2 4 Q69YZ2 TMEM200B PE2 307 1 2 5 A8MTJ6 FOXI3 PE2 420 2 2 6 P59025 RTP1 PE2 263 3 3 7 Q86WK7 AMIGO3 PE2 504 3 9 8 Q6ZU67 BEND4 PE2 534 4 6 9 Q9Y5H3 PCDHGA10 PE2 936 5 3 10 A2VCL2 CCDC162P PE2 907 6 2 11 Q6P4F1 FUT10 PE2 479 8 3 12 Q8N5D6 GBGT1 PE2 347 9 4 13 Q15760 GPR19 PE2 415 12 2 14 Q6UYE1 DLEU7 PE2 221 13 2 15 Q8N6I4 TMEM251 PE2 163 14 2 16 Q5XG99 LYSMD4 PE2 296 15 2 17 O95780 ZNF682 PE2 498 19 2 18 Q6ZMV8 ZNF730 PE2 503 19 2

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19 C9JN71 ZNF878 PE3 531 19 6 20 O75346 ZNF253 PE2 499 19 2 21 Q6ZNA1 ZNF836 PE2 936 19 3 22 Q96RD6 PANX2 PE2 677 22 4 23 P09131 SLC10A3 PE2 477 x 2 24 Q9BZD7 PRRG3 PE2 231 x 3 25 Q9UN88 GABRQ PE2 632 x 2 26 Q96MV8 ZDHHC15 PE2 337 x 4

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI.

One- TBL1Y

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in).

In collaboration with YuJu Chen, we could identify 87 MPs with one single peptide with aa ≥ 9 in subcellular fractions of hESC (Weldemariam et al., JPR SI, 2018))

Table 2- Candidate MPs (87 MPs) found in subcellular fractions of hESC (Weldemariam et al., JPR SI, 2018) with 1 peptide aa ≥ 9 Protein Protein Chr #unique Peptide # # unique No Entry Gene name Evidence length No peptides length fractions fractions 1 B7ZC32 KIF28P PE3 967 1 1 22 1 1 2 O43869 OR2T1 PE3 369 1 1 32 1 1 3 Q4KMZ8 NKAIN1 PE2 207 1 1 13 1 1 4 Q5T1S8 NCMAP PE2 102 1 1 35 2 1 5 Q8IYD2 KLHDC8A PE2 350 1 1 24 1 1 6 Q8NG84 OR2AK2 PE2 335 1 1 16 1 1 7 Q8NHC7 OR14C36 PE3 312 1 1 17 2 1 8 Q8TE23 TAS1R2 PE3 839 1 1 40 1 1 9 Q8WWF1 C1orf54 PE2 131 1 1 19 1 1 10 Q8WZ79 DNASE2B PE2 361 1 1 12 1 1 11 Q96AQ2 TMEM125 PE2 219 1 1 20 5 2 12 Q9P217 ZSWIM5 PE2 1185 1 1 24 1 1 13 A8MVX0 ARHGEF33 PE2 844 2 1 12 1 1 14 P0DJD1 RGPD2 PE2 1756 2 1 14 3 2 15 Q8NBL3 TMEM178A PE2 297 2 1 13 1 1 16 A6NFN9 ANKUB1 PE2 424 3 1 11 1 1 17 P60509 ERVPABLB-1 PE2 514 3 1 27 1 1 18 Q8IV35 WDR49 PE2 697 3 1 18 1 1 19 Q96CE8 TM4SF18 PE2 201 3 1 15 2 2 20 Q9HBL6 LRTM1 PE2 345 3 1 9 1 1 21 P18825 ADRA2C PE2 462 4 1 29 1 1 22 Q02045 MYL5 PE2 173 4 1 18 1 1 23 Q495X7 TRIM60 PE2 471 4 1 12 1 1 24 Q9BPW5 RASL11B PE2 248 4 1 20 1 1 25 A6NIZ1 NA PE2 184 5 1 27 1 1 26 A6NKW6 SHISAL2B PE3 160 5 1 13 1 1 27 Q2VIQ3 KIF4B PE2 1234 5 1 36 1 1

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28 Q8IV13 CCNJL PE2 435 5 1 11 1 1 29 Q8N9F8 ZNF454 PE2 522 5 1 16 5 3 30 Q8NGU9 GPR150 PE3 434 5 1 33 1 1 31 Q9UN71 PCDHGB4 PE2 923 5 1 9 1 1 32 Q9Y5E3 PCDHB6 PE2 794 5 1 12 1 1 33 Q9Y5H0 PCDHGA3 PE2 932 5 1 21 2 2 34 Q9Y5I1 PCDHA11 PE2 949 5 1 12 2 2 35 Q9Y5I3 PCDHA1 PE2 950 5 1 17 1 1 36 A8MW99 MEI4 PE3 385 6 1 14 2 1 37 O95047 OR2A4 PE2 310 6 1 9 1 1 38 Q96T54 KCNK17 PE2 332 6 1 10 1 1 39 Q9BZJ6 GPR63 PE2 419 6 1 10 1 1 40 Q9NUH8 TMEM14B PE2 114 6 1 29 6 2 41 Q5GH73 XKR6 PE2 641 8 1 15 1 1 42 Q6ZUA9 MROH5 PE2 1318 8 1 18 3 3 43 Q8IX29 FBXO16 PE2 292 8 1 11 1 1 44 P05015 IFNA16 PE2 189 9 1 21 1 1 45 Q5VVP1 SPATA31A6 PE3 1343 9 1 40 1 1 46 Q8NH93 OR1L3 PE2 324 9 1 26 1 1 47 O75474 FRAT2 PE2 233 10 1 20 4 3 48 Q5T5M9 CCNJ PE2 372 10 1 10 1 1 49 Q5VXJ0 LIPK PE2 399 10 1 31 1 1 50 Q86XJ0 CALHM3 PE2 350 10 1 18 1 1 51 A8MUP2 CSKMT PE2 240 11 1 19 1 1 52 Q6B0B8 TIGD3 PE2 471 11 1 11 1 1 53 Q86VR8 FJX1 PE2 437 11 1 20 2 1 54 Q8NH18 OR5J2 PE3 312 11 1 17 1 1 55 A1L157 TSPAN11 PE2 253 12 1 20 3 2 56 P46721 SLCO1A2 PE2 670 12 1 11 2 1 57 Q5BKT4 ALG10 PE2 473 12 1 10 4 2 58 Q8N1M1 BEST3 PE2 668 12 1 20 1 1 59 Q8N7H1 LINC01465 PE2 131 12 1 28 1 1 60 Q96LU7 MYRFL PE2 910 12 1 17 1 1 61 Q3B8N5 PROX2 PE2 592 14 1 12 1 1 62 Q92186 ST8SIA2 PE2 375 15 1 13 1 1 63 B3SHH9 TMEM114 PE2 223 16 1 14 2 1 64 Q9NX77 ERVK13-1 PE2 482 16 1 14 1 1 65 Q15546 MMD PE2 238 17 1 10 2 2 66 Q6QAJ8 TMEM220 PE2 160 17 1 12 3 3 67 Q6UXU6 TMEM92 PE2 159 17 1 13 4 2 68 Q8WWZ4 ABCA10 PE2 1543 17 1 16 3 2 69 A6NLF2 ELOA3D PE3 546 18 1 12 2 1 70 Q96KH6 C18orf12 PE2 178 18 1 16 1 1 71 C9JSJ3 BHMG1 PE3 638 19 1 15 1 1 72 O75373 ZNF737 PE2 536 19 1 9 2 2 73 O76099 OR7C1 PE2 320 19 1 10 1 1 74 P0C264 SBK3 PE2 359 19 1 16 2 2 75 P60606 CTXN1 PE2 82 19 1 10 1 1 76 Q8NG95 OR7G3 PE3 312 19 1 42 1 1 77 Q99742 NPAS1 PE2 590 19 1 10 1 1 78 Q9H7M6 ZSWIM4 PE2 989 19 1 17 1 1

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79 Q5W186 CST9 PE2 159 20 1 23 1 1 80 Q6ZTC4 NA PE2 211 20 1 14 1 1 81 Q9BX51 GGTLC1 PE2 225 20 1 38 1 1 82 A6NNM3 RIMBP3B PE2 1639 22 1 34 1 1 83 Q2VPJ9 LRRC75B PE2 315 22 1 21 2 1 84 O75949 FAM155B PE2 473 x 1 10 1 1 85 P78334 GABRE PE2 506 x 1 12 1 1 86 Q7Z444 ERAS PE2 233 x 1 23 1 1 87 Q96NR3 PTCHD1 PE2 888 x 1 13 1 1

G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report.

1) We showed that TBL1Y plays an important role in cardiac differentiation of human embryonic stem cell (Meyfour et al., 2017.

2) Over-expression of FUT10 (gold MP identified in our study (Weldemariam et al., JPR SI, 2018) has been reported to play a critical role in enhancement of self-renewal characteristics in embryonic stem cells (Kumar et al., J. Biol. Chem. 2013, 288, 1079 28859−28868)

Chromosome Mt PIC Leaders: Andrea Urbani Co-Pi: Paola Roncada and Mauro Fasano

Major lab members or partners contributing to the neXt50: Maurizio Ronci Luisa Pieroni Viviana Greco Vincenzo Cunsolo Salvatore Foti Federica Marini Victor C. Carregari

Status of the Chromosome “parts list” for your Chromosome:

All mitochondria encoded proteins are PE1 in nextprot. The team is currently working on PE2-5 proteins associated with mitochondria.

A) Titles and authors of papers submitted to the 2018 JPR SI and their status (eg in press, or under revision).

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Sequential Fractionation Strategy Identifies Three Missing Proteins in The Mitochondrial Proteome of Commonly Used Cell Lines. Maurizio Ronci, Luisa Pieroni, Viviana Greco, Luca Scotti, Federica Marini, Victor C Carregari, Vincenzo Cunsolo, Salvatore Foti, Antonio Aceto, Andrea Urbani. J Proteome Research, in press

Monti C, Lane L, Fasano M, Alberio T. An update of the functional mitochondrial human proteome network. J Proteome Res. 2018 Sep 19. doi: 10.1021/acs.jproteome.8b00447. [Epub ahead of print] PubMed PMID: 30230342.

B) Titles and authors of papers published in the 2017 JPR SI.

Alberio T, Pieroni L, Ronci M, Banfi C, Bongarzone I, Bottoni P, Brioschi MCaterino M, Chinello C, Cormio A, Cozzolino F, Cunsolo V, Fontana S, Garavaglia B, Giusti L, Greco V, Lucacchini A, Maffioli E, Magni F, Monteleone F, Monti M, Monti V, Musicco C, Petrosillo G, Porcelli V, Saletti R, Scatena R, Soggiu A, Tedeschi G, Zilocchi M, Roncada P, Urbani A, Fasano M. Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative. J Proteome Res. 2017 Dec 1;16(12):4319-4329. doi: 10.1021/acs.jproteome.7b00350. Epub 2017 Sep 13. PubMed PMID: 28828861.

C) Titles and authors of other HPP relevant papers submitted elsewhere in the past 2 years. Selected papers:

1: Ciregia F, Kollipara L, Giusti L, Zahedi RP, Giacomelli C, Mazzoni MR, Giannaccini G, Scarpellini P, Urbani A, Sickmann A, Lucacchini A, Bazzichi L. Bottom-up proteomics suggests an association between differential expression of mitochondrial proteins and chronic fatigue syndrome. Transl Psychiatry. 2016 Sep 27;6(9):e904. doi: 10.1038/tp.2016.184. PubMed PMID: 27676445; PubMed Central PMCID: PMC5048217.

2: Di Pierro A, Bondi H, Monti C, Pieroni L, Cilio E, Urbani A, Alberio T, Fasano M, Ronci M. Experimental setup for the identification of mitochondrial protease substrates by shotgun and top-down proteomics. EuPA Open Proteom. 2016 Feb 22;11:1-3. doi: 10.1016/j.euprot.2016.02.002. eCollection 2016 Jun. PubMed PMID: 29900104; PubMed Central PMCID: PMC5988556.

3: Fasano M, Alberio T, Babu M, Lundberg E, Urbani A. Towards a functional definition of the mitochondrial human proteome. EuPA Open Proteom. 2016 Jan 7;10:24-27. doi: 10.1016/j.euprot.2016.01.004. eCollection 2016 Mar. PubMed PMID: 29900096; PubMed Central PMCID: PMC5988588.

4: Zilocchi M, Finzi G, Lualdi M, Sessa F, Fasano M, Alberio T. Mitochondrial alterations in Parkinson's disease human samples and cellular models. Neurochem Int. 2018 Sep;118:61-72. doi: 10.1016/j.neuint.2018.04.013. Epub 2018 Apr 26. PubMed PMID: 29704589.

5: Bondi H, Zilocchi M, Mare MG, D'Agostino G, Giovannardi S, Ambrosio S, Fasano M, Alberio T. Dopamine induces mitochondrial depolarization without activating PINK1-mediated mitophagy. J Neurochem. 2016 Mar;136(6):1219-1231. doi: 10.1111/jnc.13506. Epub 2016 Jan 13. PubMed PMID: 26710242.

D) How many PE1-found MPs since HUPO-2017 has your chromosome group reported in papers?

3

E) How many PE1-found MPs since HUPO-2017 are now in NeXt-Prot as PE1 proteins? Please check each of your MPs that you reported in the JPR SI.

3

F) How many candidate MPs found, but not meeting the guidelines 2.1? (Please state number of peptides identified, their length, and biological replicates found in).

4 (please, see table hereafter)

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G) Any significant clinical or other successes re a MP that you wish us to consider highlighting in the report.

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