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Tools for Genomics, Epigenetics and Proteomics

Bioanalytical Services

Genomic Analysis Proteomic Analysis Human DNA ID
  • DNA microarray Services
  • SNP Genotyping
  • Expression Profiling
  • Nucleic Acid Isolation, Quality Check
  • Real-time PGR (qPCR)
  • Short Tandem Repeat (STR)
  • Bioin formatic & Data Analysis
  • Cell line Identification & Validation
  • Protein Identification
  • Protein Characterization
    • Peptide Mapping
    • Disulfide bond analysis
    • Glycosylation analysis
    • Glycosylation analysis
    • Protein Identification MS
  • Amino Acid Analysis
  • Protein Array Services
  • AABB accredited DNA Testing Lab
  • Paternity /Maternity
  • Family Relationship
  • Immigration - Forensic
  • Ancestry
  • Infidelity
  • ID Card
  • Cell Line Authentication
  • And More...
 
HLA Transplant Diagnostic Microarray Custom Bio Analytical Services
  • Molecular HLA Typing Technology
    • HLA SSO Typing
    • HLA SSP Typing
    • HLA Sequencing
  • Antibody-Based HLA Typing
    • HLA Protein Microarray & Immunoassays
  • PGR Based Detection Kits
  • HLA DNA SSP Typing Kits & Reagents
  • HLA Luminex Assay Development
  • Custom Oligo Microarray
  • Peptide Microarray
  • Antiobay Microarray
  • Luminex Assays and Testings
  • Array Sample Processing and Testing
  • Array Printing and Immobilization
  • Array Related Molecular Biology
  • Bioinformatic Data Analysis

Bio-Synthesis has developed sensitive analytical methods for many types oft est agents including small molecules, peptides, DNA RNA and proteins. Each of these used in many different types of biological matrices such as plasma, brain, urine, liver, heart, and fat.


LIFE SCIENCE SERVICES

Custom Products Services Biochemicals/Reagents
Custom Oligonucleotides
  • Research DNA/RNA/BNA
  • Diagnostic Oligos
  • Therapeutic Oligos
Custom Peptides
  • Research Peptides
  • Peptide for Diagnostic
  • Therapeutic Peptides
Bioconjugations

Complete bioconjugation based drug discovery and development program for pharmaceutical and biotech companies.

Antibodies Development
  • Polyclonal Antibody
  • Monoclonal Antibody
  • Assay Development
Cell Line Services
Medicinal Chemistries
DNA Identity Testing Service

AABB Accredited DNA testing laboratory for DNA identity and relationship analysis.

HLA Typing Services

High resolution and low resolution HLA typing by using Luminex SSO and PGR- SSP typing system.

Molecular Biology Services
  • Gene Synthesis
  • Cloning and [Expression
  • DNA & RNA Purification
  • Gene expression and Analysis
  • Bioinformatic Services
  • Functional Genomics and RNA
  • Antibody for Molecular Biology
  • Other Bio I analytical Services
Assay Development
  • Luminex Assay
  • HTS Assay Conversion
  • Specificity Assay
  • Secondary Assay
  • Compound Screening
DNA Based Kit Development
Ready-Peptide

Over 6,000 catalog peptides ready to use. All > 95% purity.

Ready-Antibody

30,000-1- high quality monoclonal and polyclonal antibodies from different host species applicable to all areas of biological research and drug discovery.

Sample Preparation kits
  • DNA, RNA, Protein Purification Kits
  • Protein Purification Kits •Urine Sample Preparation Kits
  • Blood/Plasma/Serum Kits
  • Sample Collection Kits
Molecular Reagents
  • DNA, RNA and Protein Markers
  • PGR Reagents
MolecularDiagnostics Kits Biochemical Reagents

Probe & Primer Design Workflow

Probe & Primer Design WorkflowSequence
Information
from NCBI or
other Database
Probe & Primer Design WorkflowDesign Probe or
Primer Manually
or by using
Design Software
Probe & Primer Design WorkflowEvaluate
Experimental
Results of
Assays

Design of BNANC Oligonucleotides

Design of BNANC Oligonucleotides Design of BNANC Oligonucleotides

Artificial oligonucleotides can be used for the design of specific DNA or RNA probes !

Model of BNANC[NH] TFO with dsDNA

Model of BNANC[NH] TFO with dsDNA
CPK model of TFO Expanded View

5’-d(TTTTTmCTTTmCTmCTmCT)-3’; T = BNANC[NH]

5’-d(GCTAAAAA GAAA GA GA GATCG)-3’

3’-d(CGATTTTT CTTT CT CT CTAGC)-5’ Target dsDNA

Major Techniques Needed

Electrophoresis

1D, 2D, native, reduced

Synthesis

Peptides, Oligonucleotides, Carbohydrates,Conjugates, etc.

Purifications

Peptide, Proteins, Oligonucleotides,Carbohydrates, Lipids, etc.: Various LC methods

Protein ID – DNA and RNA Sequencing

Sequencing (N-terminal, internal, C-terminal)

Mass Analysis

Protein Samples from Gels

1D, 2D, native, reduced

Analysis of Synthetic Peptide or Oligonucleotides – DNA/RNA/BNA

Peptides, Oligonucleotides, Conjugates, etc.

Analysis of purified samples

Peptide, Proteins, Oligonucleotides, Metabolites Various LC methods may be used

Protein ID

Sequencing (N-terminal, internal, C-terminal)

Center Dogma of Molecular Biology
by
FRANCIS CRICK
MRC Laboratory of Molecular Biology
Hills Road,
Cambridge CB2 2QH
The Center Dogma of Molecular Biology deals with the detailed residue-by-residue transfer of sequential information. It states that such Information cannot be transferred from protein to either protei or nucleic acid.
Nature Vol. 227 August 8 1970
Molecular Biology

Is there a protein (histone) code?

Molecules in the cell we would like to analyze

  • Proteins –catalyze reactions, form structures, control membrane permeability, cell signaling, recognize/bind other molecules, control gene function
  • Nucleic acids -DNA and RNA; encode information about proteins
  • Lipids - make up biomembranes
  • Carbohydrates - energy sources, energy storage, constituents of nucleic acids and surface membranes
  • Othersmall molecules - e.g. ATP, water, ions, etc.

A few types of RNAs Produced in Cells

Types of RNAs Functions
mRNAs messenger RNAs, code for proteins
rRNAs comprise ribosomes
tRNAs adaptors between mRNA and amino acids in protein synthesis
snRNAs splicing of pre-mRNAs
snoRNAs process and chemically modify rRNAs
MicroRNAs translation and mRNA degradation
Other non-coding
RNAs
telomere synthesis, X-chromo.
inactivation, protein transport
A few types of RNAs Produced in Cells

Primary Flows of Information and Substance in a Cell

Primary Flows of Information and Substance in a Cell

Genes involved in folate metabolism

Genes involved in folate metabolism
  MTHFR = methyene tetrahydrofolate transferase
  TS/TYMS = thymidylate synthase
  FS = 10-formylTHF synthase
  SHMT = serine hydroxymethttransferase
  MTHFD 5,10-methylenetetrahydrofolate dehydrogenase
  MS/MTR = methionine synthase
  MTRR = methionine synthase reductase
  BHMT = bataine hydroxymethttransferase
  DHFR = dihydrofolate reductase
  CBS = systathionine Β-synthase
  Modified from Ulrich CM, CEBP, 2000

Chromatin Immunoprecipitation Reveals Histone Acetylation State

Chromatin Immunoprecipitation Reveals Histone Acetylation State

Model for Transcriptional Repression and Activation in Yeast - I

Transcriptional Repression and Activation in Yeast - I

Model for Transcriptional Repression and Activation in Yeast - II

Transcriptional Repression and Activation in Yeast - II

 

Histone Codes Control Chromatin Condensation

 


 Chromatin Condensation


Proteomics – Why Now?

  • We can do it now !
  • Development of genome and protein sequence databases
    – Bioinformatics and Data mining software
  • Development of mass spectrometry instrumentation suitable to analyze biomolecules
    - Protein mass, Peptide mass, Peptide sequence
  • Development of analytical protein separation technology
    - IEF, 2D-SDS-PAGE, HPLC, Capillary Electrophoesis, Affinity Chromatography

Sample Preparation, Pipetting, Dissolving, Digesting etc.

Sample Preparation, Pipetting, Dissolving, Digesting

Purification of Biomolecules

Purification of Biomolecules

FPLC – AKTA Purifier
(Pharmacia, now GE-Amersham)

FPLC – AKTA Purifier

Automated HPLC

Automated HPLC

Protein Sequencing

4700 MALDI-TOF-TOF MS/MS

4700 MALDI-TOF-TOF MS/MS

Simple MALDI-TOF Voyager

Simple MALDI-TOF Voyager

Esquire LC-MS/MS

Esquire LC-MS/MS

Protein Identification Experiment

Protein Identification Experiment
  • Primary Structure Determination

    • The sequence of amino acids in a polypeptide is called its primary structure
      – Several methods exist to elucidate the primary structure of peptides

    Edman Degradation

  • Sequential cleavage and identification of N-terminal amino acids
  • Up to ~60 amino acid residues
Edman Degradation

Automated amino acid sequencing

  • One Edman degradation cycle beginning with a picomolar amount of polypeptide can be completed in
    approximately 30 minutes
    – Each cycle results in identification of the next amino acid residue in the peptide
Automated amino acid sequencing

PVDF Immobilization

PVDF Immobilization

1) SDS-PAGE, 2) Electro-blotting onto PVDF

Proteins on PVDF Membrane

Protein Characterization
by
N-terminal Sequencing

Proteins on PVDF Membrane

Ab Fragments Separation

Ab Fragments Separation

Ab Fragments after Reduction

Ab Fragments after Reduction

Analysis of MABs


SDS-PAGE
Electroblotting
N-terminal Sequencing

Membrane Transfer

Membrane Transfer

Transfer sandwich from cathode (-) to anode (+)

  • Sponge (s)
  • 3 sheets filter paper soaked in transfer buffer
  • Gel
  • Membrane
  • 3 sheets filter paper soaked in transfer buffer
  • Sponge (s)

Reducing PAGE & Electro-blotting of MAB’s

Samples were dissolved in Nupage loading buffer and reduced at 40°C for 3 h using 1 µl neat β-mercapto-ethanol

Experimental Conditions

  • Reducing SDS-PAGE: NuPAGETM

    4-12 % Bis-Tris Gradient Gel

  • Running Buffer:

    50 mM MES-Tris, 3.465 mM SDS, 1.025 mM EDTA pH 7.3, 180 to 200 V,~ 1.5 h.

  • Electroblotting:

    1 x NuPage transfer buffer (25 mM Bis-Tris, 25 mM Bicine, 1 mM EDTA, pH 7.2) + 20 % methanol, 1 h at 18 mA, Immobilon-P (PVDF membrane from Millipore),~ 1 h.

  • Staining of PVDF membrane

    15 to 30 Minutes in 50% Methanol, 10% acetic acid + comassie blue R250 (1 g/L)

  • Destaining

    10 Minutes to hours in 50% Methanol, 10% acetic acid.

Analysis of protein modifications
by
nano-spray LC-MSMS

Initiation of translation in procaryotes : a key step for regulation

Initiation of translation in procaryotes : a key step for regulation

From Laursen et al. 2005 Microbiol. Mol Biol Rev. 69:101

Initiation Factor 3, IF3

Initiation Factor 3, IF3

Garcia C, Fortier PL, Blanquet S, Lallemand JY, Dardel F

Solution structure of the ribosome-binding domain of E. coli translation initiation factor IF3. Homology with the U1A protein of the eukaryotic spliceosome J. Mol. Biol. v254, p.247-259

Initiation of translation in prokaryotes requires the formation of a complex between the messenger RNA, the 30 S ribosomal subunit and the initiator tRNA(fMet). Initiation factor IF3 binds to the 30 S ribosomal subunit and proof-reads the initiation complex, thereby ensuring the accuracy of this step. IF3 also plays a pleiotropic role in the regulation of translation, as a result of differential influences exerted on the levels of the initiation of translation of genes or groups of genes....

Analysis of protein from dried gels

Analysis of protein from dried gels

Gel bands labeled cut out and reconstituted over night in 500 ml extraction buffer (5% formic acid in 50% acetonitrile). Cellulose paper was removed. The gel bands were digested using a modified method similar to the one used by Shevchenko et al., 1996. LC-MS and LC-MS/MS analysis was performed using a Microm-LC and an LCQ deca.

translation initiation factor IF3.

1 mkggkrvqparpnrinreiraqevrltgvdgeqigivslnealekaeeagvdlveispna

61 eppvcrimdygkflyekskstkeqkeeqkviqvkeikfrpgtddgdyqvklrnlirfled

121 gdkakitlrfrgpemahqqigmevlnplrkdlcedmdlavvesfptkiegrqmimvlapk

181 kkq

translation initiation factor IF3

Mass spectrum at retention time 32.2 (lower trace, 5639). Note: A peak with a mass envelope from 1353 to 1381 was detected. The presence of these peak indicates that the KAEEAGVDLVEISPNAEPPVCR is labeled with the AMS fluorophor at the cysteine.

Characterization of Histones
ABRF PSRG Study

Purification
And
Analysis

Posttranslational Modifications of Histone H4 (Bovine)

S G R G K G G K G L G K G G A K R H R K

FT MOD_RES 1

N-acetylserine

FT MOD_RES 3

Symmetric dimethylarginine (alternate) (By similarity)

FT MOD_RES 5

N6-acetyllysine (By similarity)

FT MOD_RES 8

N6-acetyllysine (By similarity)

FT MOD_RES 12

N6-acetyllysine (By similarity)

FT MOD_RES 16

N6-acetyllysine

FT MOD_RES 20

N6,N6-dimethyllysine (alternate)

FT MOD_RES 20

N6-methyllysine (alternate)

Purification

Purification of H4 (lane3) from a commercially available histone preparation (lane 2) by ion exchange and reversed-phase chromatography

PAGE

Purification

ESI MS of Bovine Histone H4

ESI MS of Bovine Histone H4

Mass Range: 575-1300 m/z ; MaxEnt: 10-13 Kd; Zoom: 11.3-11.52 Kd

Deblocking Procedures

Procedure Number of Sites
TFA/MeOH
Bergman et al. (1996) FEBS Lett. 390, 199-202
9
GAS TFA
On-Sequencer and off-line
10
Liquid TFA
Wellner et al. (1990) Proc. Natl Acad. Sci. USA 87, 1947-1949
2
BrCN/formic acid 1

Species Identification

Three sites reported the identification of histone H4 for a particular species -> ?
Arabidopsis 1

MSGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLAR

40
C. elegans 1

MSGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLAR

40
Yeast 1

MSGRGKGGKGLGKGGAKRHRKILRDNIQGITKPAIRRLAR

40
Bovine 1

MSGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLAR

40
 
Arabidopsis 41

RGGVKRISGLIYEETRGVLKIFLENVIRDAVTYTEHARRK

80
C. elegans 41

RGGVKRISGLIYEETRGVLKVFLENVIRDAVTYCEHAKRK

80
Yeast 41

RGGVKRISGLIYEEVRAVLKSFLFSVIRDSVTYTEHAKRK

80
Bovine 41

RGGVKRISGLIYEETRGVLKVFLENVIRDAVTYTEHAKRK

80
 
Arabidopsis 81

TVTAMDVVYALKRQGRTLYGFGG

103
C. elegans 81

TVTAMDVVYALKRQGRTLYGFGG

103
Yeast 81

TVTSLDVVYALKRQGRTLYGFGG

103
Bovine 81

TVTAMDVVYALKRQGRTLYGFGG

103

Identification of Modifications

Identification of Modifications

Clostridium taeniosporum spore ribbon-1 ike appendage structure, composition and genes

James R. Walker,1* Annie J. Gnanam,1 Alexandra L. Bllnkova,1 Mary Jo Hermandson, 1F Mikhail A. Karymov,3 Yuri L. Lyubchenko,2 Paul R. Graves,38 Timothy A. Haysteac3 and Klaus D. Linse4

1 Molecular Genetics and Microbiology Section and institute for Cellular and Molecular Biology, University of Texas Austin, TX 78712. USA

2Department of Pharmaceutical Sciences, College of Pharmacy University of Nebraska Medical Center. Omaha, NE 68198. USA

3Department of Pharmacology, Duke University Medical Center, Durham, NC27710, USA.

4Protein Analysis Facility institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA.

upstream of the P29a and b genes Indicate that they likely are expressed late In the mother cell, consistent with their deposition Into the layer external to the coat.
Introduction

Endospores formed by members of the Clostridium/ Bacillus subphylum typically consist of a dehydrated core containing the chromosome, a double membrane layer, a peptidoglycan cortex, and a proteinaceous coat - struc-tures which allow spore survival of extremely harsh conditions (Driks. 2003). The principal core proteins -small acid-soluble proteins - bind and saturate the DNA (Setlow. 1966; Driks. 2002a.b). The cortex is a specialized peptidoglycan. which constricts and maintains dehydra¬tion within the core (Popham etaf.: 1995; Driks: 2003).

Appendage component
Table 2. Appendage component N-terminal and internal oligopeptide sequences.
A N-term ins I sequences"  

Present in

 
MVE LKVLXSADRS YVFFGIXN  

P29a

P29b
MR NQYLXNRNNTG/TYND  

GPS5

 
MKFVTNK  

SpoVM

 
B Internal oligopeptides in P£9a and b  

 

 
BNumber Sequence (corrected)1

Present in P29a

Present in P29b
P29-2 LDHVVFYDSLPK

+

+

P29-4

YSLSLTNIGDTK

+

-

P29-S

VFFGLSNR

+

-

P29-14 VSYSVLLTNNSNLK

+

+

P29-1E

VLTIPVIR

-

+

Internal oligopeptide in GP95

 

 

 

GPS5-2

PPGPVGPK

 

 

a. X represents an unknown residue; (GVT) could be G or T

b The PES internal peptides. 4, 5 and 14 were initially sequenced as YSLSLTTLSLP, VFFGLESS and SYSVNLTNG but corrected upon re-esannination of the raw data in light of the deduced amino acid sequences of the P£9 genes. Mess spectrometry could not distinguish I and L; the final assignments were based on the deduced amino acid sequences.

Results and Conclusions

Results and Conclusions

  • Integrated analysis via EM, direct protein sequencing of glycosylated and de-glycosylated coat proteins using Edman based as well as Mass Spectrometry based sequencing allowed to design primers for genomic sequencing.
  • A genomic gene map of the coat protein genes could be constracted.

Common Tags for Cloning and Purification of Expressed Proteins

 

His 6 aa; HHHHHH Affinity Qiagen, Invitrogen, Roche  
Step II 8 aa; WSHPQFEK Affinity IBA  
FLAG 8 aa; DYKDDDDK Immunoaffinity Sigma  
MBP 40 kDA Affinity New England Biolabs Maltose binding protein
CBP 4 kDa Affinity GE Healthcare Calmodulin binding protein epitope
TAB2 7 aa; VVSHFND   Schering AG  
Tab2s 5 aa; SHFND   Schering AG  
c-MYC 10 aa; EQKLISEEDL   Various  
HA 9 aa; YPYDVPDYA Various    
VS 14 aa; GKPIPNPLLGLDST Affinity Invitrogen  
Xpress 8 aa; DLYDDDDK Affinity Invitrogen  
BCCP       Biotin Carboxyl Carrier Protein
Nus-tag 495 aa; NusA protein Affinity Novagen  

Peptide Synthesis

Fairman`& Akerfeldt, 2005

Synthesis of acryloylated Matrix Metallo Protease Peptide Precusor for Hydrogels

Synthesis of acryloylated Matrix Metallo Protease Peptide Precusor for Hydrogels

Sep-Pak Cartridge Separation

Sep-Pak Cartridge Separation

Acryloyl-MMP purification by preparative RP-HPLC

Acryloyl-MMP purification by preparative RP-HPLC

Components of Proteomics

Components of Proteomics

Principles of MALDI-TOF Mass Spectroscopy

Principles of MALDI-TOF Mass Spectroscopy

MS Technologies

MS Technologies

Electrospray Ionization (ESI) Mass Spectrometry (MS)

Multiply charged ions of the analyte (e.g., a protein sample) are formed by protonation in an acidic solvent One, several, or many positive charges are detected Analyte is sprayed through a high-voltage nozzle into a vacuum chamber Solvent evaporate, leaving ‘naked’ ions of the multiply charged analyte Ions are detected according to mass-to-charge (m/z) ratio Detected ions are displayed as a series according to m/z ratio Computer deconvolution of the m/z peak series leads to the molecular weight of the analyte

Electrospray Ionization (ESI) Mass Spectrometry (MS)

Peptide Mass Fingerprinting:

Search m/z Mass Tolerance (Da) # Hits Database

15291 1 478
1529.7 0.1 164
1529.73 0.01 25
1529.734 0.001 4
1529.7348 0.0001 2
Effect of Mass Tolerance
1529.73 0.1 204
1529.73
1252.70
0.1 7
1529.73
1252.70
1833.88
0.1 1
Effect of Multiple Peptide Masses
The more peptides the more stringent is the ID !

Applications of Proteomics

  • Protein Mining – catalog all the proteins present in a tissue, cell, organelle, etc.
  • Differential Expression Profiling – Identification of proteins in a sample as a function of a particular state: differentiation, stage of development, disease state, response to drug or stimulus
  • Network Mapping – Identification of proteins in functional networks: biosynthetic pathways, signal transduction pathways, multiprotein complexes
  • Mapping Protein Modifications – Characterization of posttranslational modifications: phosphorylation, glycosylation, oxidation, etc.

Why is Sequence Information Important

  • A protein’s amino acid sequence is unique.
    • As little as 5 amino acid sequences can ID a protein
  • The sequence defines the primary structure of the protein
    • the primary structure is fundamental to understanding the structure and function of the protein
  • The interrelationship between an amino acid sequence and the corresponding DNA sequence
    • Protein sequences access gene sequences and are key to molecular biology
Why is Sequence Information Important

Current Methods for Proteome Research

  • SDS-PAGE
    • separates based on molecular weight and/or isoelectric point
    • 10 fmol - > 10 pmol sensitivity
    • Tracks protein expression patterns
  • Protein Sequencing
    • Edman degradation or internal sequence analysis
    • LC-MS/MS
  • Immunological Methods
    • Western Blots
Drawbacks

  • SDS-Page can track the appearance, disappearance or molecular weight shifts of proteins, but can not ID the protein or measure the molecular weight with any accuracy
  • Edman degradation requires a large amount of protein and does not work on N-terminal blocked proteins
  • Western blotting is presumptive, requires the availability of suitable antibodies and have limited confidence in the ID related to the specificity of the antibody.

Advantageous of Mass Spectrometry

  • Sensitivity in attomole range
  • Rapid speed of analysis
  • Ability to characterize and locate post-translational modifications
Papers published per year

Advantageous of Mass Spectrometry

Enzymes for Proteome Research

Trypsin
K-X and R-X except when X = P
Endoprotease Lys-C
K-X except when X = P
Endoprotease Arg-C
R-X except when X = P
Endoprotease Asp-N
X-D
Endoprotease Glu-C
E-X except when X = P
Chymotrypsin
X-L, X-F, X-Y and X-W
Cyanogen Bromide
X-M
Maldi mass spectrum
Measured (Da) Theoretical (Da) Error Residues Sequence
1381.010 1380.787 0.223 601-612 QVLLHQQALFGK
1400.884 1400.675 0.209 337-348 VVWCAVGPKKQK
1414.910 1414.752 0.158 322-333 NLRETAEEVKAR
1505.073 1505.073 0.249 302-315 IPSKVDSALYLGSR
1528.991 1528.991 0.221 337-349 VVWCAVGPEEQKK
1550.985 1550.985 0.246 630-642 NLLFNDNTECLAK
1725.122 1725.122 0.301 667-681 CSTSPLLEACAFLTR
1827.212 1827.212 0.344 379-396 GEADALNLDGGYIYTAGK

Micro-Sequencing by Tandem Mass Spectrometry (MS/MS)

  • Ions of interest are selected in the first mass analyzer
  • Collision Induced Dissociation (CID) is used to fragment the selected ions by colliding the ions with gas (typically Argon for low energy CID)
  • The second mass analyzer measures the fragment ions
  • The types of fragment ions observed in an MS/MS spectrum depend on many factors including primary sequence, the amount of internal energy, how the energy was introduced, charge state, etc.
  • Fragmentation of peptides (amino acid chains) typically occurs along the peptide backbone. Each residue of the peptide chain successively fragments off, both in the N->C and C->N direction.

Sequence Nomenclature for Mass Ladder

Sequence Nomenclature for Mass Ladder

Roepstorff, P and Fohlman, J, Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom, 11(11) 601 (1984).

 

     
Protein Sequence
GDVEKGKKIFVQK
CAQCHTVEKGGK
HKTGPNLHGLFG
R
KTGQAPGFTYT
DANKNKGITWKE
ETLMEYLENPKKY
IPGTKMIFAGIKKK
TEREDLIAYLKKAT
NE
   

Universal Proteomics Standard (UPS) Set
Catalog Number UPS1
Storage Temperature -20 °C
 
Product Description
The Universal Proteomics Standard (UPS) Set is comprised of one vial containing 48 human source or human sequence recombinant proteins {Catalog Number U6133). and one vial (20 µg) of Proteomics Grade Trypsin (Catalog Number T6567).
There are 5 pmoles of each HPLC purified protein in the vial. Each protein has been quantitated by amino acid analysis (AAA). The proteins have been selected to limit heterogeneous post-translational modifications (PTMs).
This set can be used to standardize and/or evaluate mass spectrometry (e.g., LC-MS/MS, MALDI-TOF-MS. etc.) and electrophoretic analysis conditions prior to the analysis of complex protein samples. Moreover, the set
Sigma
Components
Universal Proteomics Standard                        1 vial
5 pmoles each of 48 human proteins, dried in a 2 ml vial Catalog Number U6133
Proteomics Grade Trypsin                           20 µg
lyophilized enzyme
Catalog Number T6567

Precautions and Disclaimer
This product is for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.
Preparation Instructions
UniProt Accession number3 UniProt Protein Name [Synonym] MW (Da) (calculated) Source or recombinant Host Tag Potential PTMs*
P00709 Abha- lactalbumin 14,070 Milk     Glycosylation
P08758 Annexin A5 35,782 Placenta     Acetylation
P01008 Antithrombin-lll 49,033 Plasma     Glycosylation
P61769 Beta-2-microglobulin 11,729 Urine      
P55957 BH3 interacting domain death agonist [BID] 21,978 Recombinant E. coli    
P00915 Carbonic anhydrase 1 28,738 Erythrocytes     Acetylation
P00918 Carbonic anhydrase 2 29,095 Erythrocytes     Acetylation
P04040 Catalase 59,583 Erythrocytes      
P07339 Cathepsin D 26,624 Liver     Glycosylation
P08311 Cathepsin G 26,751 Sputum     Glycosylation
P01031 Complement C5 [Complement C5a] 8,266 Recombinant E. coli    
P02741 C-reactive protein 23,030 Plasma      
P06732 Creatine kinase M-type [CK-MM] 43,070 Heart      
POO167 Cytochrome b5 16,021 Recombinant E. coli 6-His  
P99999 Cytochrome c [Apocytochrome c] 11,60S Recombinant E. coh    
P01133 Epidermal growth factor 6,211 Recombinant E. coli    
P05413 Fatty acid-binding protein 14,716 Plasma     Acetylation Phosphorylation
P06396 Gelsolin 82,954 Plasma     Phosphorylation
P08263 Glutathione S-transferase A1[GST A1-1] 25,482 Recombinant E. coli    
P09211 Glutathione S-transferase P [GST] 23,220 Placenta      
P01112 GTPase HRas [Ras protein] 21,292 Recombinant E. coli    
P69905 Hemoglobin alpha chain 15,127 Erythrocytes      
P68871 Hemoglobin beta chain 15,867 Erythrocytes     Acetylation,
Nitrosylation,
Glycosylation
P12081 Histidyl-tRNA synthetase [Jo-1] 53.223 Recombinant E. coli    
P01344 Insulin-like growth factor II 7,464 Recombinant E. coli    
P10145 lntefleukin-8 8,381 Recombinant E. coli    
P02788 Lactotrans'errin 73,289 Miik     Glycosylation
P41159 Leptin 15,024 Recombinant E. coli    
P61626 LysozymeC 14,692 Miik      
P10636 Microtubule-associated prctein tau [Tau protein] 43,810 Recombinant      
P02144 Myoglobin 17,051 Heart      
P15559 NAD(P)H dehydrogenase [quinone] 1 [DT Diaphcrasej 33,984 Recombinant E. coli    
Q15843 Neddylin [Nedd8] 9,071 Recombinant E. coli    
P62937 Peptidyl-proiyl cis-trans isomerase A [Cyclophilin A] 17,947 Recombinant E. coli    
Q06830 Peroxiredcxin 1 22,106 Recombinant E. coli    
P01127 Platelet-derived growth factor B chain 12,286 Recombinant E. coli    
P02753 Retinol-binding protein 21,065 Unne      
P16083 Ribosyidihydronicotinannde dehydrogenase (quinone) [Quinone oxidoreductase 2 or NQ02] 25,817 Recombinant E. coli    
P02787 Serotransferrin [Apotransferrin] 75,143 Plasma     Giycosylation
P02768 Serum albjmin 65,393 Recombinant Pichia pastoris    
P63165 Small ubiquitin-reiated modifier 1 [SUMO-1] 37,420 Recombinant E. coli GST  
P00441 Superoxide dismutase [Cu-Zn] 15,800 Erythrocytes     Acetylation
P10599 Thioredcxin 12,424 Recombinant E. coli 6-His  
P01375 Tumor necrosis factor [TNF-afpha] 17,350 Recombinant E. coli    
P62988 Ubiquitn 9,387 Recombinant E. coli 6-His  
P63279 Ubiquitin-conjugating enzyme E21 [UbcH9] 17,995 Recombinant E. coli    
000762 Ubiquitin-conjugating enz/me E2 C [UbcHIO] 23,473 Recombinant E. coli 6-His  
P51965 Ubiquitin-coniuoatino enz/me E2 E1 fUbcH61 22,222 Recombinant E coli 6-His  

* As reported in UniProt. Potential PTMs have not been verified by Sigma.

The (ever expanding) Entrez System

Entrez System

Differential gel electrophoresis 2D (DIGE)

Differential gel electrophoresis