A. Query Prohormone Database

The PepShop database can be searched by UniProt accession number, gene symbol, organism name (7 species), exact amino acid sequence, and peptide monoisotopic mass with adjustable mass tolerance level. The output of this search is a list of prohormones meeting the search criteria. A list of prohormones searched using TKN1 (Protachykinin-1) as the prohormone symbol is shown below.

Match number	Symbol	Species	UniProt Accn	NCBI Gene	Number of peptides	Experimental peptides	Details	Select
1	TKN1	BOVIN	P01289	281512	5	Unknown	Detailed display
2	TKN1	RAT	P06767	24806	5	Unknown	Detailed display
3	TKN1	HUMAN	P20366	6863	5	Unknown	Detailed display
4	TKN1	CANFA	E2RF68	475239	5	Unknown	Detailed display
5	TKN1	MACMU	F6VX78	697350	5	Unknown	Detailed display
6	TKN1	MOUSE	P41539	21333	7	SwePep	Detailed display
7	TKN1	PIG	F1SF85	100525179	5	Unknown	Detailed display

Description of the results columns:

Match number:

The number of prohormones matching the search criteria.

Symbol:

The gene symbol of the matched prohormone.

Species:

Common english name of the organism.

UniProt Accn:

The primary accession number of prohormone in UniProt database.

NCBI Gene:

The primary accession number of prohormone in NCBI Gene database.

Number of peptides:

The total number of peptides in the searched prohormone sequence.

Experimental peptides:

The source of the reported peptides in prohormone sequences. The 'SwePep' denotes experimentally verified peptides in SwePep database. All other peptides are labelled as 'Unknown'.

Details:

The further details of the listed prohormone including sequence, peptides, cross-references to public databases including UniProt and NCBI, and calculated isotopic masses and isoelectric point of prohormones.

Select:

PepShop allow user to select one or more prohormones and perform cleavage site prediction (NeuroPred), pairwise sequence alignment (blastp) and mutiple sequence alignment (Muscle) to gain additional insight on prohormones and neuropeptides.




1. Prohormone Details


The Details column in above result table for TKN1 provides further information for each prohormone. The information includes: sequence of prohormone, cross-references to public databases, prohormone properties such as calculated isotopic masses and isoelectric point, and list of peptides. The detail of TKN1_MOUSE prohormone is provided below.


Details for identification: TKN1_MOUSE

Prohormone Information	Identifier TKN1_MOUSE Prohormone Symbol TKN1 Gene Symbol Tac1 Gene Name tachykinin 1 Entry name TKN1 Organism MOUSE
Cross-References	Database Accession UniProt P41539 NCBI Gene 21333 NCBI Protein NP_033337.1 UniGene Mm.410823 Ensembl ENSMUSG00000061762 Genome Database MGI:98474
Protein sequence	MKILVAVAVF FLVSTQLFAE EIDANDDLNY WSDWSDSDQI KEAMPEPFEH LLQRIARRPK PQQFFGLMGK RDADSSVEKQ VALLKALYGH GQISHKRHKT DSFVGLMGKR ALNSVAYERS AMQNYERRRK
Protein properties	Sequence length 130 Monoisotopic Mass 12957.50574 Average Mass 12965.5799 Isoelectric Point 8.943787 Number of peptides 7
Peptides	Peptide Copy No. Location Peptide Pep Len Mono Mass Aver Mass Isopoint Source Signal Peptide 1 1-19 Signal Peptide 19         TKN1_MOUSE[98-108] 1 98-108 HKTDSFVGLM G 11 1190.58051 1191.3681 6.743591 unknown TKN1_MOUSE[58-64] 1 58-64 RPKPQQF 7 899.50286 900.049 11.000793 SwePep TKN1_MOUSE[72-95] 1 72-95 DADSSVEKQV ALLKALYGHG QISH 24 2565.3238 2566.8541 5.999329 SwePep TKN1_MOUSE[111-126] 1 111-126 ALNSVAYERS AMQNYE 16 1844.84144 1846.0022 4.856873 unknown TKN1_MOUSE[20-56] 1 20-56 EEIDANDDLN YWSDWSDSDQ IKEAMPEPFE HLLQRIA 37 4418.99644 4421.7292 3.850281 unknown TKN1_MOUSE[111-118] 1 111-118 ALNSVAYE 8 865.42327 865.9391 4.59906 SwePep TKN1_MOUSE[59-69] 1 59-69 PKPQQFFGLM G 11 1248.63763 1249.4939 9.179993 unknown

Description for the prohormone details page:

Prohormone Information:

The entry name, prohormone symbol, gene symbol, gene name and common organism name to identify each prohormone.

Cross-References:

Each prohormone is further linked to UniPort, NCBI-Gene, NCBI-Protein, UniGene, Ensembl, and Genome Databases using primary accession number of prohormone in each database.

Protein sequence:

The sequence of each prohormone.

Protein properties:

The sequence length, calculated isotopic masses, isoelectric point and number of peptides in each prohormone.

Peptides:

The location in prohormone, sequence, sequence length, calculated isotopic masses, isoelectric point and source for all peptides in prohormone. Each peptide is linked to peptide database in PepShop.




2. Peptide Details


Each peptide of prohormone is further linked to peptide details page. The peptides page provides peptide name, sequence, source, and spectrum information (for SwePep peptides). An example of TKN1 prohormone peptide TKN1_MOUSE[58-64] is shown below.


Details for peptide: TKN1_MOUSE[58-64]

Peptide Information	Prohormone name Organism MOUSE Prohormone name TKN1 Location Start Location End
Source	SwePep     view spectra
Sequence	RPKPQQF
Properties	Length 7 Monisotopic Mass 899.50286 Average Mass 900.049 Isoelectric Point 11.000793 Copy number 1

Description for the entries on peptide page:

Peptide Information:

The peptide information section provides information about prohormone name, species of origin, prohormone symbol, and location of peptide in prohormone.

Source:

The source of listed peptide. The spectrum information of each peptide can be viewed though 'view spectra' link in SwePep database.

Sequence:

The sequence of each peptide.

Properties:

The sequence length, calculated isotopic masses, isoelectric point, and number of occurrences of each peptide in prohormone.




3. SwePep Information


The spectrum information of each SwePep peptide can be viewed using view spectra link on peptides detail page. The SwePep page provides list of all spectra reported for the selected peptide. The SwePep information of TKN1_MOUSE[58-64] is shown below.


Description of columns on SwePep page:

log(e):

The log-transformed expectation value of X!Tandem.

m+h:

The observed mass of peptide.

delta:

The difference between observed and calculated mass of each peptide.

z:

The peptide ion charge state.

peptide:

The sequence of peptide and its location in prohormone sequence.



The peptide column links to spectrum page that provides information about prohormone, plot of m/z vs. intensity of each MS/MS peak, and a table of observed b and y ions. An example is shown below. The spectrum of TKN1_MOUSE[58-64] peptide is shown below.



B. Predict Fragment Ions from Sequence


The program predict fragment ions predicts the fragment ion-series and there mass-to-charge (m/z) ratio according to the provided options. The submitted sequence is cleaved at all specified bonds (option: ion types) along the peptide backbone and the resultant N-termianl and C-terminal ions are displayed upto provided maximum precursor charge state. An example output of KYVMGHFRWD peptide is shown below. Among other parameters: precursor charge state:+2, mass type:monoisotopic, ion types:b and y ions, and both ammonia and water losses were set to yes.


Peptide Ion

+1MH	+2MH	Mass Type	Sequence
1338.6416	669.8247	Mono	KYVMGHFRWD

Fragment Ions

ION	+1MH	+2MH	+1MH-18	+2MH-18	+1MH-17	+2MH-17	ION	+1MH	+2MH	+1MH-18	+2MH-18	+1MH-17	+2MH-17
b1	129.1028	65.0553	--	--	112.0763	56.5421	y9	1210.5466	614.7825	1192.5361	605.7772	1193.5201	606.2692
b2	292.1661	146.587	--	--	275.1396	138.0737	y8	1047.4833	533.2508	1029.4728	524.2456	1030.4568	524.7376
b3	391.2345	196.1212	--	--	374.208	187.6079	y7	948.4149	483.7166	930.4044	474.7114	931.3884	475.2034
b4	522.275	261.6414	--	--	505.2485	253.1281	y6	817.3744	418.1964	799.3639	409.1911	800.3479	409.6831
b5	579.2965	290.1522	--	--	562.27	281.6389	y5	760.3529	389.6856	742.3424	380.6804	743.3264	381.1724
b6	716.3554	358.6816	--	--	699.3289	350.1684	y4	623.294	321.1562	605.2835	312.1509	606.2675	312.6429
b7	863.4238	432.2158	--	--	846.3973	423.7026	y3	476.2256	247.622	458.2151	238.6167	459.1991	239.1087
b8	1019.5249	510.2664	--	--	1002.4984	501.7531	y2	320.1245	169.5714	302.114	160.5662	--	--
b9	1205.6042	603.306	--	--	1188.5777	594.7928	y1	134.0452	76.5318	116.0347	67.5265	--	--

Description of columns in peptide and fragment ion tables:

Peptide Ion-⁺¹MH and ⁺²MH:

The precursor mass at charge states +1 and +2, respectively.

Mass Type:

The isotope used in calculating the precursor mass value.

Sequence:

The sequence of peptide submitted by the user.

Fragment Ions-ION:

Type of ions selected by the user.

Fragment Ions-⁺¹MH, ⁺¹MH^-18, and , ⁺¹MH^-17:

The mass values of fragment ion (b and y ions), mass values of fragment ions after water loss, and mass values after ammonia losses at charge state +1, respectively. The rest of the columns represent mass values at increasing charge states.

C. MS/MS spectral search

Description of MS/MS searh results:

Search engine:

The database search program used to identify peptide from MS/MS data.

Search title:

The search title entered at the start of search by user.

MGF file:

The Mascot Generic Format (MGF) file with MS/MS data provided as input by the user.

Number of peptides assigned:

The total number of peptides assigned by the database search program to the input spectrum.

Search Paramters:

The search paramters specified by the user. More detail about these parameters is avaiable in the input section.

Peptide Name:

The name assigned to the peptide sequence in PepShop database. Each peptide identification is linked with peptide detail page in PepShop peptide database providing additional information about peptides.

Calc. Mass:

The calculated mass of the peptide.

Length:

The peptide sequence length.

E-value:

The expectation value assigned by OMSSA program to peptide identification. The E-value represents the expected number of database matches by chance with scores equal or higher than the one observed. Lower E-value denotes more significant peptide identification.

Sequence:

The sequence of the peptide.

Prohormone:

Primary accession number of prohromone is UniProt database. This accession number links each identified peptide with prohormones in PepShop prohormone database.

Isoelectric Point:

The calculated isoelectric point of the identified peptide.

Database Status:

The source of peptide in PepShop peptide database.

D. NeuroPred

1. Cleavage Prediction Diagram

2. Mass of Predicted Peptides

Description of the columns for the Mass of Predicted Peptides table

Abb. Peptide:

Abbreviated Peptide where the start and the end of the peptide is provided by a single letter amino acid code and location within the sequence.

NCut:

The model or models that predicted cleavage at this site that corresponds to the start or N-terminal region of the peptide. Signal Peptidase or Sequence Start are used to denote the start of the peptide whether a signal peptide is indicated or not.

CCut:

The model or models that predicted cleavage at this site that corresponds to the end or C-terminal region of the peptide. Sequence End is used to denote the end of the sequence.

PTM Applied:

Provides which PTMs have been applied to the peptide. Most PTM designations are self-explanatory; however, "Cleaved" denotes that the peptide has only been cleaved, "Extended" denotes that adjacent peptides have been joined, and "TrimKR" indicates that all the C-terminal K and R amino acids have been removed after cleavage. When a PTM is applied multiple times, only the mass with all occurrences is reported and the frequency is also reported in the "PTM Applied" column by indicating the number of PTM occurrences, followed by the times symbol ("x"), (e.g., Acetylation 3x).

Predicted Aver. Mass:

Predicted average mass of the peptide including any PTMs.

Predicted Mono. Mass:

Predicted monoisotopic mass of the peptide including any PTMs.

Peptide Sequence:

Complete sequence of the peptide. Any post-translational modifications that have been successfully applied are reported using small blue brackets (e.g. PQQFFGLM[Amide] denotes amidation of the peptide PQQFFGLM).

E. Multiple Sequence Alignment Using Muscle

TKN1_BOVIN      MKILVAvAViFfiSTQLsAEEIGANDDfNYWSDWSDSDQIKEEmPEPFEHLLQRIARRPK
TKN1_HUMAN      MKILVALAVFFLVSTQLFAEEIGANDDLNYWSDWyDSDQIKEELPEPFEHLLQRIARRPK
TKN1_CANFA      MKILVALAVFFLVSTQLFAEEIGANDDLNYWSDWSDSDQIKEELPEPFEHLLQRIARRPK
TKN1_MACMU      MKILVALAVFFLVSTQLFAEEIGANDDLNYWSDWSDSDQIKEELPEPFEHLLQRIARRPK
TKN1_PIG        MKILVALAVFFLVSTQLFAEEIGANDDLNYWSDWSDSDQIKEELPEPFEHLLQRIARRPK
TKN1_MOUSE      MKILVAvAVFFLVSTQLFAEEIdANDDLNYWSDWSDSDQIKEamPEPFEHLLQRIARRPK
TKN1_RAT        MKILVAvAVFFLVSTQLFAEEIGANDDLNYWSDWSDSDQIKEamPEPFEHLLQRIARRPK

TKN1_BOVIN      PQQFFGLMGKRDADSSIEKQVALLKALYGHGQlSHKRHKTDSFVGLMGKRALNSVAYERS
TKN1_HUMAN      PQQFFGLMGKRDADSSIEKQVALLKALYGHGQISHKRHKTDSFVGLMGKRALNSVAYERS
TKN1_CANFA      PQQFFGLMGKRDA---------------GHGQISHKRHKTDSFVGLMGKRALNSVAYERS
TKN1_MACMU      PQQFFGLMGKRDADSSIEKQVALLKALYGHGQISHKRHKTDSFVGLMGKRALNSVAYERS
TKN1_PIG        PQQFFGLMGKRDADSSIEKQVALLKALYGHGQISHKRHKTDSFVGLMGKRALNSVAYERS
TKN1_MOUSE      PQQFFGLMGKRDADSSvEKQVALLKALYGHGQISHKRHKTDSFVGLMGKRALNSVAYERS
TKN1_RAT        PQQFFGLMGKRDADSSIEKQVALLKALYGHGQISHKRHKTDSFVGLMGKRALNSVAYERS

TKN1_BOVIN      vMQdYERRRK
TKN1_HUMAN      AMQNYERRR-
TKN1_CANFA      AMQNYERRRK
TKN1_MACMU      AMQNYERRR-
TKN1_PIG        AMQNYERRRK
TKN1_MOUSE      AMQNYERRRK
TKN1_RAT        AMQNYERRRK

F. Local Pairwise Sequence Alignment Using Blastp

BLASTP 2.2.24+


Reference:
Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schäffer,
Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997),
"Gapped BLAST and PSI-BLAST: a new generation of protein database
search programs", Nucleic Acids Res. 25:3389-3402.



Reference for composition-based statistics:
Alejandro A. Schäffer, L. Aravind, Thomas L. Madden, Sergei
Shavirin, John L. Spouge, Yuri I. Wolf, Eugene V. Koonin, and
Stephen F. Altschul (2001), "Improving the accuracy of PSI-BLAST
protein database searches with composition-based statistics and
other refinements", Nucleic Acids Res. 29:2994-3005.



Database: prohormones
           668 sequences; 118,462 total letters



Query=  TKN1_MOUSE SignalP=19
Length=130
                                                                      Score     E
Sequences producing significant alignments:                          (Bits)  Value

  TKN1_MOUSE |symbol=TKN1;GeneSymbol=Tac1;GeneName=tachykinin 1;G...   271    1e-75
  TKN1_RAT |symbol=TKN1;GeneSymbol=Tac1;GeneName=tachykinin, prec...   268    1e-74
  TKN1_PIG |symbol=TKN1;GeneSymbol=Tac1;GeneName=tachykinin 1;Gen...   263    3e-73
  TKN1_MACMU |symbol=TKN1;GeneSymbol=TAC1;GeneName=tachykinin, pr...   261    9e-73
  TKN1_HUMAN |symbol=TKN1;GeneSymbol=TAC1;GeneName=tachykinin, pr...   259    6e-72
  TKN1_BOVIN |symbol=TKN1;GeneSymbol=Tac1;GeneName=tachykinin 1;G...   251    9e-70
  TKN1_CANFA |symbol=TKN1;GeneSymbol=Tac1;GeneName=tachykinin 1;G...   225    6e-62
  TKN4_HUMAN |symbol=TKN4;GeneSymbol=TAC4;GeneName=tachykinin 4 (...  28.9    0.013
  TKN4_CANFA |symbol=TKN4;GeneSymbol=Tac4;GeneName=tachykinin 4;G...  28.1    0.022
  TKN4_MOUSE |symbol=TKN4;GeneSymbol=Tac4;GeneName=tachykinin 4;G...  26.9    0.041
  TKN4_BOVIN |symbol=TKN4;GeneSymbol=Tac4;GeneName=tachykinin 4;G...  26.9    0.043
  TKNK_BOVIN |symbol=TKNK;GeneSymbol=Tac2;GeneName=tachykinin 2;G...  26.6    0.066
  TKNK_CANFA |symbol=TKNK;GeneSymbol=Tac2;GeneName=tachykinin 2;G...  26.2    0.073
  TKNK_RAT |symbol=TKNK;GeneSymbol=Tac2;GeneName=tachykinin 2;Gen...  25.4    0.12 
  TKN4_PIG |symbol=TKN4;GeneSymbol=Tac4;GeneName=tachykinin 4;Gen...  25.4    0.12 
  TKNK_MOUSE |symbol=TKNK;GeneSymbol=Tac2;GeneName=tachykinin 2;G...  25.4    0.12 
  TKNK_HUMAN |symbol=TKNK;GeneSymbol=TAC3;GeneName=tachykinin 3;G...  25.4    0.12 
  TKN4_RAT |symbol=TKN4;GeneSymbol=Tac4;GeneName=tachykinin 4 (he...  25.4    0.12 
  TKNK_MACMU |symbol=TKNK;GeneSymbol=TAC3;GeneName=tachykinin 3;G...  25.0    0.15 
  TKNK_PIG |symbol=TKNK;GeneSymbol=Tac2;GeneName=tachykinin 2;Gen...  25.0    0.17 
  TRH_MOUSE |symbol=TRH;GeneSymbol=Trh;GeneName=thyrotropin relea...  21.9    1.5  
  GON2_PIG |symbol=GON2;GeneSymbol=NA;GeneName=NA;GeneID=nomatch;...  21.9    1.6  
  PNOC_BOVIN |symbol=PNOC;GeneSymbol=Pnoc;GeneName=prepronocicept...  19.6    7.1  
  CORT_RAT |symbol=CORT;GeneSymbol=Cort;GeneName=cortistatin;Gene...  19.2    10.0