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Mass Spectrometry – Sample Preparation
136 To place an order call your local office or visit sigma.com/order.sigma.com/proteomics
Trypsin, Proteomics GradeTrypsin Singles, Proteomics Grade
Trypsin Spin Column, Proteomics Grade
Catalog Number T6567 T7575 TT0100
Package Sizes 5 × 20 µg (sufficient to digest 2-10 mg of sample)
96 × 1 µg (sufficient to digest 96 samples, 20-100 µg of each)
10 columns (sufficient to digest 10 samples, 10-100 µg each)
Features n Reductively methylated to minimize autolytic activity
n TPCK treated to quench chymotryptic activity
n Extensively purified
n All the advantages of Proteomics Grade Trypsin in a convenient, single-use 1 µg package
n Eliminates repetitive pipetting
n 15-minute protein digestionn Eluted peptides are ready for MS
analysis – no additional sample preparation required
Components n Trypsin, Proteomics Grade n Trypsin, Proteomics Grade (T6567)n Trypsin Solubilization Buffern Enzyme Reaction Buffer
n Trypsin Spin Columns containing 75 mg of solid support in a 50% acidic glycerol suspension
n Collection Tubesn Enzyme Reaction Buffer
Suggested Sample Size 20-100 µg of sample per 1 µg of trypsin 20-100 µg of sample per 1 µg of trypsin single
10-100 µg of sample per spin column
Digest Time Overnight Overnight 15 minutes
Digest Types Solution or In-Gel Solution or In-Gel Solution
Enzyme Purity >98% >98% N/A
Chymotryptic Activity None Detected None Detected None Detected
Prequalified Downstream Applications
HPLC-MS, MALDI-MS HPLC-MS, MALDI-MS HPLC-MS, MALDI-MS
Trypsin and Protease Selection Guide
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Trypsin Profile In-Gel Digest Kit Protease Profiler 18O Proteome Profiler
PP0100 PP0500 (Individual components also available separately, see below.)
P3623
1 kit (sufficient for up to 100 excised protein spots) 1 kit (sufficient to digest up to 5,900 µg of sample) 1 kit (sufficient for 12 experiments)
n Fast, efficient, complete in-gel tryptic digestion from PAGE to MALDI
n Digested proteins are ready for MALDI-MS – no additional sample preparation required
n Five proven proteases for detailed characterization of proteins of interest
n Perform double enzymatic digestsn Explore alternative proteases
n Simplest, most efficient method for relative MS quantitation of differential expression
n Efficient incorporation of stable isotopes (>98%)n Fewer autolytic peptides than the competition
n Trypsin, Proteomics Grade (T6567)n Destaining Solutionn Enzyme Reaction Buffern Biotechnology Grade Acetonitrilen Trypsin Solubilization Reagentn Peptide Extraction Solution
n Trypsin, Proteomics Grade (T6567)n Asp-N Protease (P3303)n Lys-C Protease (P3428)n Glu-C Protease (P6181)n Arg-C Protease (P6056)n Enzyme Solubilization Reagentn Enzyme Reaction Buffer
n 18O water, 99% Isotopic Purityn Trypsin, Proteomics Grade (T6567)n Trypsin Singlesn Trypsin Solubilization Reagentn Enzyme Reaction Buffern Microspin Columns and Collection Tubesn Biotechnology Grade Acetonitrilen 1.0% TFA Solutionn Siliconized Microcentrifuge Tubes, 0.6 mln 1 cc Syringesn 23 Gauge Needles
One excised protein spot or band n 20–100 µg of sample per 1 µg of Trypsinn 50–200 µg of sample per 1 µg of Asp-Nn 20–100 µg of sample per 1 µg of Lys-Cn 20–100 µg of sample per 1 µg of Glu-Cn 20–100 µg of sample per 1 µg of Arg-C
2 × 50 µg
Overnight Overnight 2 days
In-Gel Solution or In-Gel Solution
>98% 95–98% >98%
None Detected None Detected None Detected
HPLC-MS, MALDI-MS HPLC-MS, MALDI-MS HPLC-MS, MALDI-MS
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138 To place an order call your local office or visit sigma.com/order.sigma.com/proteomics
Trypsin Modification
Trypsin
Lysine Residue
Chymotrypsin
Porcine trypsin contains 10 Lysine (K) residues;each represents a potential site for autodigestion.Chymotrypsin and other contaminant proteins are
often present as well.
Total dimethylation of the -amino groups of lysine protects trypsinfrom autodigestion.
TPCK bindsirreversibly to the activesites of any chymotrypsinmolecules present,rendering them inactive.
Affinitypurification removescontaminants, improving activity and preventing ambiguous MALDI spectra.
Proteomics Grade Trypsin is dimethylated at eachLysine (K) residue, preventing autodigestion. Any residual
inactivated Chymotrypsin molecules and other minor contaminants have been removed.
Contaminants
Methyl groups
TPCK
Proteomics Grade TrypsinSuperior Sequence Coverage Without Ambiguous Peaks!
Proteomics Grade Trypsin shows excellent proteolytic efficiency, generating more tryptic peptides leading to greater sequence coverage of your protein of interest. Mass spectra are significantly simplified due to the reduced number of interfering autolytic peaks and their ambiguous adducts. Proteomics Grade Trypsin has been extensively purified from porcine pancreas to enable accurate and precise cleavage on the carboxylic side of Arg and Lys residues. The enzyme has been exhaustively processed by reductive methylation to minimize autolysis and chymotryptic activity has been quenched by TPCK (N-Tosyl-L-phenylalanine chloromethyl ketone) treatment. Further purification steps including affinity chromatography and lyophilization from dilute acid produce a highly purified, high specific activity trypsin purposely suited for the demanding criteria of proteomics research, and designed to function in either solution or gel based digests. The enzyme is conveniently packaged in 20 mg vials to ensure fresh enzyme is available for each use.
In-Gel Trypsin Digestion of 20 pmoles of Carbonic Anhydrase IIR
elat
ive
Inte
nsi
ty
800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
0
20
40
60
80
1000
20
40
60
80
100 Competitor A
Competitor B
A7
T9
T16
T1
T3
T2
T13 T7
T5
T14 T7-8
A14
T9
T1
T3
T2
T T1312-13
T7
T5
T14T8-9
A3
A4
0
20
40
60
80
100
A7
T9
T16
T1 T3 T2
T13 T7
T5
T14
T7-8
T4
T10T22
T20
T2
-
Sigma Trypsin
1
Sigma’s Proteomics Grade Trypsin leads to greater sequence coverage than other trypsins. To compare trypsin from Sigma and two other suppliers, a sequence coverage experiment was performed. In each instance, 20 pmoles of carbonic anhydrase was run on a 10% tris-glycine SDS-PAGE gel. The gel was stained with EZBlue™ Gel Staining Reagent (G1041). Following destaining, the bands were excised and tryptically digested with the Trypsin Profile In-Gel Digest Kit (PP0100). MALDI analysis was performed in the reflectron positive ion mode using α-cyano-4-hydroxycinamic acid (10 mg/ml in 70% ACN, 0.03% TFA) as the matrix. The sample was mixed 1:1 with the matrix and 1 ml was spotted onto the MALDI target. Thirteen tryptic fragments (up to nearly 2,900 M+H+) of carbonic anhydrase and one autolytic fragment were identified using Sigma Proteomics Grade Trypsin; only ten tryptic fragments and one autolytic fragment were identified using competitor A’s trypsin, and ten tryptic fragments and three autolytic fragments were identified using competitor B’s trypsin.
Ordering InformationCat. No. Product Description Quantity
T6567 Trypsin, Proteomics Grade 5 × 20 µg
T7575 Trypsin Singles, Protemics Grade 96 × 1 µg
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Components
Proteomics Grade Trypsin
Destaining Solution
Trypsin Reaction Buffer
Biotech Grade Acetonitrile
Trypsin Solubilization Reagent
Peptide Extraction Solution
Trypsin Profile IGD Kit Workflow
Excise protein of interest
Transfer liquidto a new tube
Destain gel slice
Place gel slice in siliconized tube
Remove destaining solution and dry gel in SpeedVac®
Rehydrate with Trypsin solution
Combine liquid with previous extract
Incubate for 6 hrs to overnight at 37 ºC
Optional:
Add peptideextractionsolution to gel slice
Incubate 30 min. at 37 ºC
Sample is readyfor MALDI-MS
Trypsin Profile IGD KitIn-gel tryptic digests of 1D or 2D acrylamide gels are commonly used in proteomics to identify unknown proteins of interest. The unknown proteins are excised from the gel, digested with trypsin, and then analyzed via MALDI or HPLC-MS with subsequent database searching. Sigma’s Trypsin IGD Kit provides a quick and easy method for the digestion of up to 100 samples. Trypsin IGD Kit contains Proteomics Grade Trypsin and is optimized for polyacrylamide gels stained with Coomassie® or SYPRO® Orange or Ruby dyes. If silver stained gels are used, a different destaining step is required (ProteoSilver™ Plus, Silver stain-ing kit is recommended, Cat. No. PROTSIL2). A destaining solution for silver stained gels is included in the ProteoSilver Plus kit and the gel pieces should be destained according to the included protocol.
Identification of E. coli Protein
0
20
40
60
80
100
Rel
ativ
e In
ten
sity
Mass/Charge
1000 1500 2000 2500 3000 3500800 4000
T10T10
*
T19
T21-22
T20
T20-21
T9
T15
T13
-
-
The sample was desalted using a C18 pipette tip and eluted directly onto the MALDI target using the MALDI matrix (α-cyano-4-hydroxycinnamic acid, 10 mg/ml in 70% ACN, 0.03% TFA). MALDI analysis was performed in the reflection positive ion mode. The resulting monoisotopic masses were searched against the NCBI database at a tolerance of 150 ppm. The protein was identified as outer membrane protein 3a from E. coli with the matched peptides providing 30% sequence coverage.
Ordering InformationCat. No. Product Description Quantity
PP0100 Trypsin Profile IGD Kit 1 kit
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Mass Spectrometry – Sample Preparation
140 To place an order call your local office or visit sigma.com/order.sigma.com/proteomics
Trypsin Spin Column, Proteomics GradeRevolutionize Your Mass Spectrometry Sample Prep
Traditional tryptic digests can require up to 18 hours; Sigma’s NEW Trypsin Spin Column can complete the same digest in only 15 minutes!
The ultra-micro spin column contains highly purified, TPCK treated porcine trypsin immobilized on a chemically modified silica support to minimize non-specific adsorption. Eluted peptides are ready for MS analysis with no additional clean-up.
Features and Benefitsn Reduce tryptic digest time to 15 minutes
n Successfully digest and recover 10 to 100 µg of protein sample
n Minimize autolytic and chymotryptic spectral peaks
n Analyze samples directly following digestion – no clean-up required
From 18 hours to 15 minutes: Reduce Tryptic Digest Time and Achieve Excellent Results
In-solution
18 hours
Spin Column
15 minutes
Protein Substrate In Solution Spin Column Digest Time: 18 hours Digest Time: 15 minutes
b-Galactosidase 13% 8 peptides 10% 6 peptides
BSA 14% 15 peptides 46% 21 peptides
Fumerase 8% 2 peptides 56% 18 peptides
Carbonic Anhydrase 18% 3 peptides 34% 5 peptides
Lysozyme 68% 6 peptides 44% 4 peptides
Sequence coverage and number of peptides identified by MALDI-TOF-MS. A five-protein mixture was digested through a standard 18-hour protocol and with the Trypsin Spin Column. Both digests were analyzed by MALDI-TOF-MS and the sequence coverage of each protein calculated. In 4 out of 5 cases, the sequence coverage obtained with the spin column digest was similar to or better than that obtained from the solution digest. The proteins were reduced, alkylated, and digested in urea. The sequence coverage for fumarase and carbonic anhydrase was significantly enhanced with the spin column digestion.
Ordering InformationCat. No. Product Description Quantity
TT0010 Trypsin Spin Column, Proteomics Grade* 10 columns
(*sufficient to digest 10 to 100 µg of sample per column)
Components
Trypsin Spin Columns, containing 75 mg (150 ml) of solid support in a 50% acidic glycerol suspension, 10 each
Collection Tubes, 20 each
Enzyme Reaction Buffer, 25 ml
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MiniTip™ C18 Micropipette TipsSuperior Recovery, Exceptional Binding
Expertly designed by chromatographers and mass spectrometrists, the MiniTip C18 pipette tip provides superior sample recovery, increased bed integrity, and enhanced binding capacity for purifying and concentrating mass spectrometry samples. MiniTips’ unique, proprietary adhesive chemistry greatly increases available silica particle surface area, leading to exceptional binding capacity and unsurpassed analyte recovery. The unique solid phase support binds peptides, proteins, and other analytes retained by reversed phase techniques while withstanding the rigors of repeated draw and dispense cycles without structural degradation. For optimal purification and concentration of important samples, trust MiniTips!
Superior RecoveryMass spectrometrists are often concerned that important samples may be lost during concentration and purification. To address this concern, Sigma MiniTips have been designed to provide the most efficient recovery available. MiniTips’ proprietary adhesive chemistry and optimum adhesive to silica ratio maximize the available silica surface area, which is nearly three times that of competitors. This increased surface area enables greater C18 binding and greater affinity for protein and peptide samples. During repeated draw and dispense cycles, more sample is retained by the MiniTip, and more sample is subsequently recovered. In an experiment evaluating MiniTips and two competitors, peptide samples were purified, eluted, and analyzed by MALDI-MS. Signals for the samples prepared using MiniTips were greatly enhanced when compared to compet-ing tips. MiniTips also perform more consistently than alternative tips. In the experiment described, MiniTips exhibited an average relative standard deviation of 14%. Competitor M’s average relative standard deviation was 73% and Competitor E’s average relative standard deviation was 94%.
More Efficient Recovery, Greater Reproducibility Peptide Signal After Purification from 400 µM Urea
0
200
400
600
800
1000
1200
Bradykinin Angiotensin II P14R
Inte
nsi
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Sigma MiniTip Competitor M Competitor E
MiniTip Workflow
Wet the tip with70% Acetonitrile
Equilibrate the tip with0.1% Formic Acid
Elute analyte in matrix
Sample in >20 mlof aqueous solvent
Wash awaysuppressing ions
Bind analyte through repeated pipetting
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Enhanced Affinity, Exceptional Binding CapacitySigma MiniTips consistently demonstrate both improved analyte affinity and superior binding capacity when compared to other tips. MiniTips’ greater silica surface area leads to additional C18 binding and increases affinity for proteins and peptides. As analytes are drawn through the C18 silica, they are retained more readily and in greater quantities than in competitors’ tips. Enhanced affin-ity ensures that even low abundance samples are retained and purified.
MiniTips saturate with analyte at a much higher concentration than other tips, enabling purification of larger samples. Increased binding capacity is particu-larly beneficial when analysis conditions require more sample. In an experiment using b-amyloid, MiniTips demonstrated nearly twice the binding capacity of an alternative tip.
MALDI Analysis with and without MiniTips
500.3208
550.5271
634.2198
804.1765
1038.5484
1085.4230
1281.6137
1272.5017
1507.6250
1930.7919
1888.8273
1890.7514 2057.8356
2642.9208
Before Cleanup with MiniTips C18
Protein Not Identified
Post Cleanup with MiniTips C18
Signal Enhanced, Protein Identified
Stable Bed Minimizes Degradation and Contamination Even under the most demanding conditions, the design of the MiniTip prevents sorbent bed degradation. While other tips collapse under standard conditions, leaving contaminating material in valuable samples, the MiniTip maintains its integrity. A Sigma MiniTip and a competitor’s C18 tip were used to purify and concentrate a protein sample. Upon coloration of the silica within the tips, it was evident that competitor M’s sorbent bed was broken and fragmented while that of the MiniTip remained intact, demonstrating its integrity.
Ordering InformationCat. No. Product Description Quantity
TPSC18 MiniTip C18 96 tips 960 tips
Bulk quantities available.
b-Amyloid Binding Capacity (µg)
0.0
Sigma MiniTip Replicates Competitor M Replicates
5.0
10.0
15.0
20.0
18.7218.18
19.34
8.667.80
8.52
Competitor M after use.
MiniTip after use.
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Proteome Profiler 18O Enzymatic Labeling Kit Discover improved expression profiling with the enzymatic incorporation of a stable 18O isotope label. In the presence of trypsin, oxygen atoms from H2
18O will specifically exchange with the two oxygen atoms of the carboxyl terminus of each tryptic peptide. The incorporation of two 18O atoms results in a 4 Da mass shift, which is readily observed when analyzed by mass spectrometry. The mass shift allows labeled and unlabeled samples to be pooled and analyzed for differential expression.
Convenient, efficient, and affordable, the Proteome Profiler 18O Enzymatic Labeling Kit provides the reagents, labware, and an optimized method required to perform this time-tested strategy. Well characterized and trusted, 18O labeling is the proven solution for your global proteome profiling needs.
MALDI-TOF Mass Spectrum
1420
100
90
80
70
60
50
40
30
20
10
01425 1430 1435
1432.6778
1433.6977
1428.6384
1429.6204 1434.6814
1435.7020
1430.6392
1431.6583
Mass/Charge1440 1445
Theoretical Ratio 1:3Observed Ratio 1:2.9
MALDI-TOF mass spectrum of the peptide FESNFNTQATNR, derived from lysozyme (hen egg white). This represents expected results obtained when two protein solutions, with concentra-tion ratios of 1:3, based on protein amounts, are analyzed using this kit. The base peak (a) at m/z 1428.65 (mass + 0 Da) is one third of the integrated peak area, as the peak (b) at m/z 1432.68 (mass + 4 Da). The peaks at 1429.62, 1430.64, and 1431.66, as well as those at 1433.70 and 1434.68, represent the normal isotopic distribution obtained by the mass spectrometric analysis of a peptide. The observed ratio was determined to be 2.93.
Ordering InformationCat. No. Product Description Quantity
P3623 Proteome Profiler 18O Enymatic Labeling Kit* 1 kit
(*sufficient for 12 experiments)
Components18O water, 99% Isotopic Purity, 550 µl
Trypsin, Proteomics Grade, 2 × 20 µg
Trypsin Singles, 24 × 2 µg
Trypsin Solubilization Reagent, 1 ml
Enzyme Reaction Buffer, 2 × 25 ml
Microspin Columns & Collection Tubes, 24 each
Acetonitrile, 1 ml
1.0% TFA, 1 ml
Siliconized Microcentrifuge, Tubes, 0.6 ml, 50 each
1 cc. Syringe, 12 each
23 Gauge Needle, 12 each
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Mass Spectrometry – Sample Preparation
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Components
Trypsin, Proteomics Grade (T6567)
Asp-N Protease (P3303)
Lys-C Protease (P3428)
Glu-C Protease (P6181)
Arg-C Protease (P6056)
Enzyme Solublization Reagent
Enzyme Reaction Buffer
Protease Profiler KitIdentify and characterize your unknown proteins with accuracy and convenience using the Protease Profiler Kit.
Proteolytic digestion of an unknown sample protein is often used to determine or confirm its identity. Proteases with specific, optimal cleavage sites generate predictable, consistent peptide products, simplifying data interpretation. Trypsin is recognized as the most commonly used protease for protein identification – given a favorable primary structure and tertiary conformation, trypsin is an excellent choice. In some cases, trypsin may cleave an active site, or cleavage sites may be inaccessible; an alternative protease is needed. The Protease Profiler Kit provides four excellent alternative proteases, in addition to Proteomics Grade Trypsin, an enzyme solubilization reagent, and enzyme reaction buffer. The Profiler Kit enables optimized, convenient digestion of target proteins with a variety of prequalified, well-established proteases.
Enzyme Specificity Optimal pH Average MW
Proteomics Carboxyl side of pH 8.0 23.3 kDa Grade arginine and Trypsin lysine residues
Asp-N Amine side of pH 6.0-8.5 24.5 kDa Aspartic and Cysteic acid residues
Glu-C Carboxyl side of pH 4.0-7.8 29.0 kDa glytamyl and aspartyl residues
Lys-C Carboxyl side of pH 8.5 28.0 kDa Lysine residues
Arg-C Carboxyl side of pH 7.5-8.5 26.5 kDa Arginine residues, as well as Lys-Lys
Ordering InformationCat. No. Product Description Quantity
PP0500 Protease Profiler Kit 1 kit
T6567 Proteomics Grade Trypsin 5 × 20 µg
P3303 Asp-N Protease 2 µg
P6181 Glu-C Protease 25 µg
P3428 Lys-C Protease 5 µg
P6056 Arg-C Protease 5 µg
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ProteoMass™ Guanidination KitDramatically improve sensitivity, increase sequence coverage and identify with greater confidence!
Boost your MALDI-MS sensitivity with the NEW ProteoMass Guanidination Kit! Peptides generated during proteolytic digestion with C-terminal Arginine residues are ionized preferentially over peptides with C-terminal Lysine residues, leading to compromised sequence coverage and limited confidence during peptide mass fingerprint analysis. The ProteoMass Guanidination Kit efficiently and conveniently converts C-terminal Lysine residues to Homoarginine, increas-ing MALDI signal strength and producing enhanced sequence coverage.
Features and Benefitsn Identify more samples with greater accuracy and confidence
n Increase throughput and save time – only 35 minutes to use the kit vs. 2 hours using traditional methods
n Compatibility – compatible with 1D or 2D PAGE gel bands or spots, as well as complex cell extracts
Enhanced Ionization, Improved Signal to Noise
2950 2960 2970 2980 2990 3000Mass/Charge
Mass Shift +42
%Int.
100
90
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70
60
50
40
30
20
10
0 3010
MALDI-TOF mass spectrum of the control peptide TNEIVEEQYPTQSLATLESQELGK*. This spec-trum represents a 1:1 mixture of guanidinated and unguanidinated peptide.
Guanidination Reaction
+
O
OH
NH2
N
H
R
C-terminal lysine
H2N
CH3
NHC
O
O-Methylisourea
NH2
O
OH
NH
NH
N
H
R
Homoarginine
Ordering InformationCat. No. Product Description Quantity
MS0100 ProteoMass Guanidination Kit 1 kit
Components
O-Methylisourea hemisulfate
Base reagent
Stop Solution
Control Peptide
Guanidination Kit Workflow
Adjust pHand mix
Trypticpeptides
Analyze by MALDI;observe enhancedsignal
Add stop solutionand mix
Add guanidinationreagent and mix
Incubate at 65 ˚C
Store samplesat 2-8 ˚C
Addmatrix
Total timeonly 35 minutes
Mass Spectrometry – Expression Analysis & Quantitation
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AQUA Peptides™
Focus! With Sigma, focus your protein biomarker research!
The AQUA TechnologyDeveloped in collaboration with the Protein Quantitation Consortium, Sigma’s AQUA Peptides enable accurate, efficient mass spectrometric quantitation of protein biomarkers.
n Focus your analysis on significant protein biomarkers
n Accurately quantitate low abundance proteins
n Eliminate costly, time-consuming stable isotope labeling steps
n Measure site-specific phosphorylation states
n Validate gene silencing at the protein level
The Protein AQUA StrategyIn June 2003, Dr. Steve Gygi and his team presented an innovative strategy, Protein AQUA, enabling absolute protein quantitation using stable isotope labeled peptides and HPLC-MS. By applying a common principle, the use of a labeled molecule as an internal standard, to protein analysis, Gygi’s team has advanced the abilities of protein researchers to study complex biological samples quantitatively and has provided a valuable new tool for Proteomics.
An AQUA Peptide is simply a synthetic tryptic peptide corresponding to a peptide of interest. Each AQUA peptide incorporates one stable isotope labeled amino acid, creating a slight increase (6-10 Daltons) in molecular mass. When mixed, the endogenous peptide and the synthetic AQUA Peptide elute together chromatographically, ionize with the same intensity, and fragment identically. However, the endogenous peptide and the synthetic AQUA Peptide are easily distinguished in a mass spectrometer by their molecular masses.
In a typical AQUA experiment, a known amount of AQUA Peptide is added to a biological protein sample. The sample is then digested and analyzed by HPLC-MS. Extracted ion chromatograms are generated for the endogenous peptide and the synthetic AQUA Peptide internal standard. Using peak ratios, the quantity of endogenous peptide is calculated.
Protein-AQUA is a powerful, enabling technology, the limits of which are only now being explored. For proteomics researchers, it facilitates focused, quantitative studies of not only specific protein expression, but specific amino acid modification as well.
This method was developed by Dr. Steve Gygi and colleagues at Harvard Medical School [Stemmann O, Zou H, Gerber SA, Gygi SP, Kirschner MW; Dual inhibition of sister chromatid separation at metaphase, Cell 2001, Dec 14, 107: 715-726]. Limited use of this method ispermitted under a licensing arrangement with Harvard Medical School. Protein-AQUA andAQUA Peptide are trademarks of Harvard Medical School.
AQUA Workflow
Select an optimal trypticpeptide and stable isotopeamino acid from the sequence of your protein of interest
Order synthetic AQUA Peptidefrom Sigma-Genosys
Optimize LC-MS/MS separation protocol for quantitation
Step 1: AQUA Peptide Selection
VPQVSTPTLVEVSR
VPQVSTPTLVEVSR*
Extract protein from biological samples and add known quantity of AQUA Peptide
Digest
* Labeled amino acid
Analyze by LC-MS/MS or MALDI to quantitate protein of interest
[ ]
Step 2: Implementation
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The AQUA Peptide™ Partnership Program and LibrarySigma is pleased to announce the launch of its AQUA Peptide Partnership Program and the upcoming debut of the AQUA Peptide Library. With over 400 library peptides currently in development, the AQUA Peptide Library promises even greater convenience and value for quantitative proteomics research.
We invite you, the proteomics community, to contribute to the AQUA Peptide Library through the AQUA Peptide Partnership Program. When you order custom AQUA Peptides, you will be invited to add your sequences to the library. Contributors will receive partnership pricing on their custom orders. The larger proteomics community will enjoy the benefit of accessing the sequences quickly and conveniently in the following months.
Peptides in the AQUA Peptide Library will be available to purchase in the coming weeks, with new peptides being added rapidly. We look forward to working with you to build the library together!
To learn more about the AQUA Peptide Partnership Program, visit sigma.com/aqua.
Custom AQUA PeptidesTo meet the specific demands of AQUA experimentation, Sigma has developed a specialized custom peptide offering. Custom AQUA Peptides are synthesized using fully labeled 98 atom% 13C and 98 atom% 15N enriched amino acids (one labeled amino acid per peptide) and are stringently tested to ensure high purity (HPLC), accurate molecular weight (ESI-MS), and specific peptide content.
To order Custom AQUA Peptides, call 1- 800-325-3010 or visit sigma.com/aquaorder
Ordering InformationProduct Name Quantity
Custom AQUA Peptide 5 × 1 nmol
Available AQUA Amino Acids
Stable Isotope Labeled Mass Difference between Amino Acid (Fully Labeled, Native Peptide and 13C, 15N > 98 atom %) AQUA Peptide
l-Arginine-13C6,15N4 10 Daltons
l-Isoleucine-13C6,15N 7 Daltons
l-Leucine-13C6,15N 7 Daltons
l-Lysine-13C6,15N2 8 Daltons
l-Phenylalanine-13C9,15N 10 Daltons
l-Proline-13C5,15N 6 Daltons
l-Valine-13C5,15N 6 Daltons
AQUA Library Peptidesn >95% pure by HPLC
n Quantitated by AAA
n Confirmed by ESI-MS
n Individual 100 pmol packages
n Available immediately
Custom AQUA Peptides n >95% pure by HPLC
n Quantitated by AAA
n Confirmed by ESI-MS
n 5 × 1 nmol packages
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148 To place an order call your local office or visit sigma.com/order.sigma.com/proteomics
Selecting the Best AQUA PeptideSelecting an optimal AQUA Peptide is key to your analytical success. Because your time and resources are valuable, we suggest a careful evaluation of candidate peptides. The following guidelines provide a good starting point. Using the known specificity of trypsin and the sequence of your protein of interest, generate a list of all of its tryptic peptides. Using the guidelines below, eliminate unsuitable peptides and select peptides with optimal characteristics for AQUA experimentation.
n Choose a peptide that is unique to your protein of interest
n Choose a tryptic peptide which resolves well by HPLC. Avoid peptides that are too hydrophilic or too hydrophobic
n Choose a peptide which ionizes well in your MS system
n Avoid chemically reactive residues such as Tryptophan, Methionine, Cysteine, or chemically unstable sequences such as Asp-Gly, N-term Gln, and N-term Asn
n Choose an amino acid from the available list to be isotopically labeled
n Peptide sequence length should be limited to 15 amino acids or less
Verify Gene SilencingVerification of gene silencing represents a novel and powerful application of the AQUA technique. AQUA quantitation is an effective method for measur-ing expression of silenced genes and verifying knockdown success. AQUA quantitation offers many advantages over traditional methods for measuring expression of silenced genes.
n AQUA sensitivity is limited only by the sensitivity of the mass spectrometer, enabling researchers to study gene silencing in low abundance proteins, or to detect very low expression of silenced genes. This represents a marked advantage over traditional methods of Western blotting and ELISA
n An AQUA Peptide can be generated using only an amino acid sequence, even when no native peptide has been isolated or when no corresponding antibody exists. For this reason, AQUA can be used to measure any silenced gene while alternative methods, such as Western blotting, cannot
n AQUA quantitation measures protein (and corresponding peptide) expression. This provides a more accurate correlation to silencing efficiency when compared to quantitative PCR or Northern blotting
Learn more at sigma.com/aquagene
GAPDH Measured with AQUA
350
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50
0Mock
Non-interfe
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RISC free
GAPDH (9.6 nM)
GAPDH (19 nM)
GAPDH (38 nM)
pm
ol G
APD
H
HeLa cultures transfected with siRNA for Human GAPDH (9.6 nM, 19 nM, and 38 nM), RISC free (non-functional, non-interfering), two non-interfering sequences, and a non-interfering pool. Additionally, four mock transfections were performed. Transfected cells were incubated 48 additional hours. Proteins were extracted, precipitated, quantitatively spiked with AQUA peptides, tryptically digested, and analyzed by reversed phase LC-MS. Restricted ion current chromatograms for native and AQUA peptides from GAPDH were generated; based on peak area ratios, GAPDH concentrations were calculated.
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149Our Innovation, Your Research — Shaping the Future of Life Science
Increase the Sensitivity of Protein AQUA by Depleting High Abundance ProteinsThe study of the Plasma Proteome has historically been fundamentally limited by its vast dynamic range (10 orders of magnitude). Low abundance proteins, carrying great diagnostic potential, are often obscured by the presence of high-abundance serum proteins. By combining Sigma’s ProteoPrep® 20 Plasma Technology and the Protein AQUA technology, scientists can both identify and quantitate numerous lower-abundance proteins in human serum. This combination of technologies may provide a targeted approach to the discovery of proteins holding biological and diagnostic significance.
Protein-AQUA Analysis of Gelsolin in Whole Plasma and ProteoPrep 20 Depleted Samples. SRM Analysis of the endogenous gelsolin peptide, GASQAGAPQGR, results in a complex chromatogram in whole plasma (A) with poor resolution and low signal-to-noise. When analyzed in depleted plasma (C), the endog-enous gelsolin peptide is easily separated from interfering species and its concentration readily calculated by comparison to the AQUA peptide internal standard (B and D).
Learn more at sigma.com/aquadepletion
Protein AQUA Analysis of Gelsolin in Whole Plasma and ProteoPrep 20 Depleted Samples
A
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RT: 26.78
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RT: 26.82
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Gelsolin Peptide in
Depleted Plasma
Gelsolin Peptide in Plasma
Endogenous
AQUA AQUA
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RT: 26.82
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Endogenous
Gelsolin Peptide in
Depleted Plasma
Gelsolin Peptide in Plasma
Endogenous
AQUA AQUA
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RT: 26.78
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25.69 28.5123.38
33.1731.96
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RT: 26.81
AA: 10369
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RT: 26.68
AA: 4753
RT: 26.82
AA: 133133
Endogenous
Gelsolin Peptide in
Depleted Plasma
Gelsolin Peptide in Plasma
Endogenous
AQUA AQUA
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25.69 28.5123.38
33.1731.96
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RT: 26.81
AA: 10369
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RT: 26.68
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RT: 26.82
AA: 133133
Endogenous
Gelsolin Peptide in
Depleted Plasma
Gelsolin Peptide in Plasma
Endogenous
AQUA AQUA
C
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RT: 26.78
AA: 270
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25.69 28.5123.38
33.1731.96
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RT: 26.81
AA: 10369
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RT: 26.68
AA: 4753
RT: 26.82
AA: 133133
Endogenous
Gelsolin Peptide in
Depleted Plasma
Gelsolin Peptide in Plasma
Endogenous
AQUA AQUA
22 24 26 28 30 32 34
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RT: 26.78
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29.58
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30.87
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25.69 28.5123.38
33.1731.96
27.44
RT: 26.81
AA: 10369
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RT: 26.68
AA: 4753
RT: 26.82
AA: 133133
Endogenous
Gelsolin Peptide in
Depleted Plasma
Gelsolin Peptide in Plasma
Endogenous
AQUA AQUA
D
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RT: 26.78
AA: 270
29.58
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31.01
30.87
31.14
25.69 28.5123.38
33.1731.96
27.44
RT: 26.81
AA: 10369
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RT: 26.68
AA: 4753
RT: 26.82
AA: 133133
Endogenous
Gelsolin Peptide in
Depleted Plasma
Gelsolin Peptide in Plasma
Endogenous
AQUA AQUA
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Time (min)
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RT: 26.78
AA: 270
29.58
26.30
29.18
31.01
30.87
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25.69 28.5123.38
33.1731.96
27.44
RT: 26.81
AA: 10369
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RT: 26.68
AA: 4753
RT: 26.82
AA: 133133
Endogenous
Gelsolin Peptide in
Depleted Plasma
Gelsolin Peptide in Plasma
Endogenous
AQUA AQUA
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Mass Spectrometry – Expression Analysis & Quantitation
150 To place an order call your local office or visit sigma.com/order.sigma.com/proteomics
Enhance Sensitivity of Protein AQUA of Phosphopeptides through Enrichment with a Novel Gallium-IMAC Spin ColumnThe analysis of phosphorylated peptides by mass spectrometry has often been limited by their intrinsically low abundance and poor ionization. An effective strategy to both enhance phosphopeptide MS signal levels and quantitate specific phosphopeptides involves using Protein AQUA. Using a Gallium Immobilized Metal Affinity Chromatography (IMAC) based microspin column and phosphorylated AQUA Peptides, scientists can easily enrich, identify, and quantitate specific phosphopeptides under varying biological conditions.
EIC of AQUA Phosphopeptides
Load Enriched
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8.76
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10012.09
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A B
390 pg load
3.9 ng load
39 ng load
390 pg load
3.9 ng load
39 ng load
Extracted ion currents of AQUA phosphopeptides contained in the load and elution fractions from the Phosphopeptide Enrichment Kit (Cat. No. PP0410). The sample load prior to enrich-ment (A) demonstrates significant noise levels with little signal. Following IMAC enrichment of 39 ng, 3.9 ng, and 390 pg loads, the enriched (elution) fractions appropriately show decreasing signal levels of the phosphopeptide (B). Further analysis of the 390 pg column load by SRM indicated that the phosphopeptide was observable with an acceptable signal-to-noise level and would allow for quantitation using the AQUA methodology.
Learn more at sigma.com/aquaphospho
Analytical Strategy
Peptides Phosphopeptides
Complex protein mixture
Endogenous protein digestAQUA phosphopeptide
Non-phosphorylated AQUA peptide
PhosphopeptideEnrichment
Flow-throughfraction
Elutedfraction
TrypticDigest
Workflow highlighting the use of Phosphopeptide Enrichment Kit for selective enrichment of the phosphorylated species.
SRM of AQUA Phosphopeptide
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SRM of phosphopeptide
SRM development of AQUA phosphopeptides to examine low levels of phosphorylation within a biological sample. The sensitivity of SRM facilitates detection of a 390 pg IMAC load.
Iden
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151Our Innovation, Your Research — Shaping the Future of Life Science
Stable Isotope Labeling with Amino Acids – SILACStable isotope labeling with amino acids in cell culture (SILAC), has recently gained popularity for its ability to compare the expression levels of hundreds of proteins in a single experiment. SILAC was developed at CEBI1 as a simple and accurate approach for MS-based quantitative proteom-ics. The method relies on the incorporation of unlabeled and D, 13C, and/or 15N-labeled amino acids added to the growth media of separately cultured cell lines, giving rise to cells containing either “light” or “heavy” proteins, respectively. Upon mixing lysates collected from these cells, digests of the protein populations are analyzed by mass spectrometry. Using this method, one can compare the expression levels for hundreds of proteins.
This valuable and robust proteomics tool is being used in many different applications. Here are a few examples:
n Biomarker discovery2
n Peptide turnover kinetics3
n Complete proteome analysis4
References1. Ong, S.E., et al., Mol Cell Proteomics 1, 376–86, (2002). 2. Gronborg, M., et al., Mol Cell Proteomics 5, 157–171, (2006). 3. Milner E., Barnea E., Beer I, and Admon, A., Mol Cell Proteomics
5: 357–365, (2006). 4. de Godoy, L.M., Olsen J.V., de Souza G.A., Li G., Mortensen P., Mann
M., Genome Biol, 7(6): R50, (2006).
SILAC Workflow
Culture A“Light” Amino Acid(ex. L-Arginine HCl)
rel.
inte
nsit
y
m/z
Culture B“Heavy” Amino Acid
(ex. L-Arginine-13C6 HCl)
Coming Soon
DMEM and RPMI 1640 deficient media for SILAC
For stable isotope bulk inquiries, please contact Isotec at (800) 448-9760 • (937) 859-1808 or [email protected].
Primary “Light” Amino Acids
Cat. No. Description
A6969 l-Arginine HClI7403 l-Isoleucine L8912 l-LeucineL8662 l-Lysine HClM5308 l-MethionineT8441 l-ThreonineT8566 l-TyrosineV0513 l-Valine
Additional Products of InterestF0392 Fetal Bovine Serum DialyzedG7513 l-Glutamine solution P4333 Penicillin-Streptomycin solution (100×) StabilizedT3924 Trypsin-EDTA solutionL5750 Sodium dodecyl sulfateS2127 Sodium carbonate 09830 Ammonium bicarbonate I6125 Iodoacetamide S8157 Silver nitrate A6283 Acetic acid, glacialM3641 Methanol B2025 Brilliant Blue G - Colloidal Concentrate
Primary “Heavy” Amino Acids
Cat. No. Description
600113 l-Arginine-15N4 HCl608033 l-Arginine-13C6, 15N4 HCl643440 l-Arginine-13C6 HCl608092 l-Isoleucine-13C6, 15N486825 l-Leucine-5,5,5-d3
608068 l-Leucine-13C6, 15N605239 l-Leucine-13C6
608041 l-Lysine-13C6, 15N2 HCl616192 l-Lysine-4,4,5,5-d4 HCl643459 l-Lysine-13C6 HCl299154 l-Methionine-13C,d3 (methyl-13C,d3) 300616 l-Methionine-d3 (methyl-d3)608106 l-Methionine-13C5, 15N608149 l-Methionine-1-13C,d3 (carboxy-13C, methyl-d3)607770 l-Threonine-13C4, 15N489794 l-Tyrosine-13C6 (phenyl-13C6)492868 l-Tyrosine-13C9
607991 l-Tyrosine-13C9, 15N600148 l-Valine-13C5, 15N
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Mass Spectrometry – Standards & Calibrants
152 To place an order call your local office or visit sigma.com/order.sigma.com/proteomics
ProteoMass Peptide & Protein Calibration Kit ProteoMass Peptide Calibration Kit
Catalog Number MSCAL1 MSCAL2
Package Sizes 1 kit 1 kit
Components Calibrantsn Bradykinin Fragment 1–7, 10 nmolsn Angiotensin II, 10 nmolsn P14R, 10 nmolsn ACTH Fragment 18–39, 10 nmolsn Insulin Oxidized B-chain, 10 nmolsn Insulin, 10 nmolsn Cytochrome c, 10 nmolsn Apomyoglobin, 10 nmolsn Aldolase, 10 nmolsn Albumin, 10 nmols
Matricesn α-cyano, 5 × 10 mgn sinapinic acid, 5 × 10 mg
Solventsn 0.1% TFAn 1% TFAn Acetonitrile
Calibrantsn Bradykinin Fragment 1–7, 2 × 10 nmolsn Angiotensin II, 2 × 10 nmolsn P14R, 2 × 10 nmolsn ACTH Fragment 18–39, 2 × 10 nmolsn Insulin Oxidized B-chain, 2 × 10 nmols
Matrix n α-cyano, 10 × 10 mg
Solventsn 0.1% TFAn 1% TFAn Acetonitrile
Primary Application(s) Calibration and sensitivity testing of MALDI-MS instruments in mass ranges from 757 Daltons to 66,000 Daltons.
Calibration and sensitivity testing of MALDI-MS instruments in mass ranges from 757 Daltons to 3,500 Daltons.
Unique Attributes & Advantages
n Stringently assayed to ensure MALDI-MS performance and compatibility
n Free of salts and metal ions, improving ionization efficiency and avoiding generation of ambiguous spectral adducts
n Conveniently packaged, freeing you from time-consuming purification and weigh-up steps, allowing you to focus on acquisition of quality data
n Stringently assayed to ensure MALDI-MS performance and compatibility
n Free of salts and metal ions, improving ionization efficiency and avoiding generation of ambiguous spectral adducts
n Conveniently packaged, freeing you from time-consuming purification and weigh-up steps, allowing you to focus on acquisition of quality data
ProteoMass™ Calibrant Selection Guide
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153Our Innovation, Your Research — Shaping the Future of Life Science
ProteoMass Protein Calibration Kit ProteoMass vMALDI™ Calibration Kit ProteoMass Guandination Kit
MSCAL3 MSCAL4 MS0100
1 kit 1 kit 1 kit
Calibrantsn Insulin, 2 × 10 nmolsn Cytochrome c, 2 × 10 nmolsn Apomyoglobin, 2 × 10 nmolsn Aldolase, 2 × 10 nmolsn Albumin, 2 × 10 nmols
Matrixn sinapinic acid, 10 × 10 mg
Solventsn 0.1% TFAn 1% TFAn Acetonitrile
Calibration Mix, Standard Rangen 5 vials, each containing MRFA, Bradykinin 1–7,
Angiotensin 1, Neurotensin, Renin Substrate, and Bradykinin
Calibration Mix, High Rangen 5 vials, each containing MRFA, Bradykinin,
ACTH 1–16, Melittin, and ACTH 7-38
Sensitivity Standardn Angiotensin II
Matrixn α-cyano, 2 × 10 mg
Solventsn 1% TFAn Acetonitrilen Ethanol, 200 Proof, Molecular Biology Grade
n O-Methylisourea hemisulfaten Base reagentn Stop Solutionn Control Peptide
Calibration and sensitivity testing of MALDI-MS instruments in mass ranges from 5,700 Daltons to 66,000 Daltons.
Calibration and sensitivity testing of the Thermo-Fisher LTQ™-MS with vMALDI ion source instrument in mass ranges from 523 Daltons to 3,675 Daltons.
Enhancement of MALDI signal strength and sequence coverage through efficient conversion of C-terminal Lysine residues to Homoarginine.
n Stringently assayed to ensure MALDI-MS performance and compatibility
n Free of salts and metal ions, improving ioniza-tion efficiency and avoiding generation of ambiguous spectral adducts
n Conveniently packaged, freeing you from time-consuming purification and weigh-up steps, allowing you to focus on acquisition of quality data
n Developed and qualified specifically for use with the Thermo-Fisher LTQ-MS with vMALDI ion source
n Contains high purity, low alkali metal solvents and recrystallized matrix
n Conveniently packaged, freeing you from time-consuming purification and weigh-up steps, allowing you to focus on acquisition of quality data
n Identify more samples with greater accuracy and confidence
n Increase throughput and save time – only 35 minutes required
n Compatible with 1D or 2D PAGE gel bands or spots, as well as complex cell extracts
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Mass Spectrometry – Standards & Calibrants
154 To place an order call your local office or visit sigma.com/order.sigma.com/proteomics
The Universal Proteomics Standard and the Proteomics Dynamic Range Standard are a complex, well-defined, well-characterized reference standard for mass spectrometry. Both standards contain the same 48 human proteins (6,000 to 83,000 Daltons). Each constituent protein has been HPLC purified and AAA quantitated prior to formulation.
Universal Proteomics Standard UPS1Developed in collaboration with the Association of Biomolecular Resource Facilities (ABRF) Proteomics Standards Research Group (sPRG), the Universal Proteomics Standard contains 48 human proteins (5 pmoles of each).
Proteomics Dynamic Range Standard UPS2This standard is an enhancement of the Universal Proteomics Standard (UPS1). The same complex mixture of 48 human proteins has been formulated into a dynamic range of concentration levels, ranging from 50 pmoles to 0.5 fmoles.
Features and Benefitsn Test the power of your analytical strategy
n Troubleshoot and optimize your analytical protocol
n Confirm system suitability before analyzing critical samples
n Normalize analytical results day-to-day or lab-to-lab
Ordering InformationCat. No. Product Description Quantity
UPS1 Universal Proteomics Standard Set* 1 set
UPS2 Proteomics Dynamic Range Standard Set* 1 set
* Each set contains one vial of Universal Proteomics Standard and one vial (20 µg) of Proteomics Grade Trypsin
For more information, visit sigma.com/ups
UniProt Accession Number3
UPS1 Amount (fmol)
UPS2 Amount (fmol) UniProt Protein Name (Synonym)
MW (Da) (calculated)
P00915 5,000 50,000 Carbonic anhydrase 1 28,738P00918 5,000 50,000 Carbonic anhydrase 2 29,095P01031 5,000 50,000 Complement C5 [Complement C5a] 8,266P69905 5,000 50,000 Hemoglobin alpha chain 15,127P68871 5,000 50,000 Hemoglobin beta chain 15,867P41159 5,000 50,000 Leptin 16,024P02768 5,000 50,000 Serum albumin 66,393P62988 5,000 50,000 Ubiquitin 9,387P04040 5,000 5,000 Catalase 59,583P00167 5,000 5,000 Cytochrome b5 16,021P01133 5,000 5,000 Epidermal growth factor 6,211P02144 5,000 5,000 Myoglobin 17,051P15559 5,000 5,000 NAD(P)H dehydrogenase [quinone] 1
[DT Diaphorase]30,984
P62937 5,000 5,000 Peptidyl-prolyl cis-trans isomerase A [Cyclophilin A]
17,947
Q06830 5,000 5,000 Peroxiredoxin 1 22,106P63165 5,000 5,000 Small ubiquitin-related modifier 1
[SUMO-1]37,420
P00709 5,000 500 Alpha-lactalbumin 14,070P06732 5,000 500 Creatine kinase M-type [CK-MM] 43,070P12081 5,000 500 Histidyl-tRNA synthetase [Jo-1] 58,223P61626 5,000 500 Lysozyme C 14,692Q15843 5,000 500 Neddylin [Nedd8] 9,071P02753 5,000 500 Retinol-binding protein 21,065P16083 5,000 500 Ribosyldihydronicotinamide
dehydrogenase (quinone) [Quinone oxidoreductase 2 or NQO2]
25,817
UniProt Accession Number3
UPS1 Amount (fmol)
UPS2 Amount (fmol) UniProt Protein Name (Synonym)
MW (Da) (calculated)
P63279 5,000 500 Ubiquitin-conjugating enzyme E2 I [UbcH9] 17,995P01008 5,000 50 Antithrombin-III 49,033P61769 5,000 50 Beta-2-microglobulin 11,729P55957 5,000 50 BH3 interacting domain death agonist [BID] 21,978P07339 5,000 50 Cathepsin D 26,624P08263 5,000 50 Glutathione S-transferase A1 [GST A1-1] 25,482P01344 5,000 50 Insulin-like growth factor II 7,464P01127 5,000 50 Platelet-derived growth factor B chain 12,286P10599 5,000 50 Thioredoxin 12,424P08311 5,000 5 Cathepsin G 26,751P99999 5,000 5 Cytochrome c [Apocytochrome c] 11,608P06396 5,000 5 Gelsolin 82,954P09211 5,000 5 Glutathione S-transferase P [GST] 23,220P01112 5,000 5 GTPase HRas [Ras protein] 21,292P02787 5,000 5 Serotransferrin [Apotransferrin] 75,143O00762 5,000 5 Ubiquitin-conjugating enzyme E2 C
[UbcH10]20,473
P51965 5,000 5 Ubiquitin-conjugating enzyme E2 E1 [UbcH6]
22,222
P08758 5,000 0.5 Annexin A5 35,782P02741 5,000 0.5 C-reactive protein 23,030P05413 5,000 0.5 Fatty acid-binding protein 14,716P10145 5,000 0.5 Interleukin-8 8,381P02788 5,000 0.5 Lactotransferrin 78,289P10636 5,000 0.5 Microtubule-associated protein tau [Tau
protein]46,810
P00441 5,000 0.5 Superoxide dismutase [Cu-Zn] 15,800P01375 5,000 0.5 Tumor necrosis factor [TNF-alpha] 17,350
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155Our Innovation, Your Research — Shaping the Future of Life Science
Calibration Workflow
Peptide 110 l
100 l
Peptide 210 l
100 l
Peptide 310 l
Dissolve desiredpeptides in solventto 100 pmole/ml, vortex.
Combine 10 mlof each peptide.
Add 70 ml solventto make the finalvolume 100 ml
Dissolve matrixin solvent to 10 mg/ml, vortex.
Combine equalvolumes peptide mixture and matrix.
Apply 1 ml to MALDItarget and dry.
100 l
70 l solvent
1 ml solvent
10 mg matrix
10 l
10 l
1 l
MALDITarget
ProteoMass™ MALDI Calibration KitsThe most flexible kits for routine MS calibration.The expanding field of proteomics involves many unique aspects, particularly in terms of specific reagent needs for MALDI-MS analysis – Sigma understands and meets these needs. The ProteoMass family of products includes conveniently packaged kits, highly purified matrices and solvents, and a wide range of well-recognized, pre-qualified calibrants. Great care is taken to certify the absence of salts and metal ions, improving the ionization efficiency of species of interest and avoiding the generation of ambiguous spectral adducts. Specialized packaging eliminates potential introduction of contaminant oils, ions, or other substances. Corrosive solvents are provided ready-to-use to minimize handling and promote safety. Calibrate, tune, and test sensitivity with ProteoMass MALDI Calibration Kits and individual reagents – the answers to your MALDI-MS reagent needs!
PSD of P14R
2000 400 600 800 1000 1200 1400Mass/Charge
%Int.
b2
b3 b3
b5
b6
b7
y 7
y 8
y 9y 6
y 11
y 12
y 13
y 14
y 10
y 5-N
H3
y 6-N
H3
y 7-N
H3
y 8-N
H3
y 7-N
H3
y 10-
NH
3
y 11-
NH
3
P 14R100
90
80
70
60
50
40
30
20
10
0
X5
Post source decay analysis of P14R using α-cyano-4-hydroxycinnamic acid as the MALDI matrix. Data was acquired on a Kratos Analytical AXIMA-CFR system™ in reflectron positive ion mode.
Peptide Calibration
1000 1500 2000 2500 3000 3500Mass/Charge
%Int.
Bradykinin fragment 1-7(M+H)+
Angiotensin II(M+H)+
ACTH fragment 18-39(M+H)+
Insulin oxidized B Chain(M+H)+
100
90
80
70
60
50
40
30
20
10
0
MALDI mass spectrum of a peptide calibration solution containing 1.5 mM Bradykinin fragment 1-7, 1.0 mM Angiotensin II, 0.5 mM ACTH fragment 18-39, and 2.0 mM Insulin oxidized B chain. A 10 ml aliquot of the peptide solution in 0.1% TFA was mixed with 10 ml of a 10 mg/ml α-cyano-4-hydroxycinnamic acid in 50% ACN, 0.05% TFA. 0.8 ml of the resulting solution was spotted onto the MALDI target. Analysis was done using a Kratos Kompact SEQ system™ in the linear positive ion mode.
Ordering InformationCat. No. Product Description Quantity
MSCAL1 ProteoMass Peptide & Protein MALDI-MS 1 kit Calibration Kit
MSCAL2 ProteoMass Peptide MALDI-MS Calibration Kit 1 kit
MSCAL3 ProteoMass Protein MALDI-MS Calibration Kit 1 kit
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Mass Spectrometry – Standards & Calibrants
156 To place an order call your local office or visit sigma.com/order.sigma.com/proteomics
ProteoMass vMALDI™ Calibration KitThe ProteoMass vMALDI Calibration Kit enables calibration and sensitivity testing of the Thermo-Fisher LTQ™-MS with vMALDI ion source instrument in mass ranges from 523 to 3,675 Daltons.
Ordering InformationCat. No. Product Description Quantity
MSCAL4 ProteoMass vMALDI Calibration Kit 1 kit
ProteoMass Calibrants (Available separately or in ProteoMass Kits)Also Included In
Cat. No. Description (M+H)+ Package Size
ProteoMass Protein &
Peptide Kit (MSCAL1)
ProteoMass Peptide Kit (MSCAL2)
ProteoMass Protein Kit (MSCAL3)
B4181 Bradykinin Fragment 1–7 757.3997 (monoisotopic) 5 × 10 nmols X X
A8846 Angiotensin II (human sequence)
1,046.5423 (monoisotopic) 5 × 10 nmols X X
P2613 P14R 1,533.8582 (monoisotopic) 5 × 10 nmols X X
A8346 ACTH Fragment 18–39 (human sequence)
2,465.1989 (monoisotopic) 5 × 10 nmols X X
I6154 Insulin Oxidized B-chain (bovine)
3,494.6543 (average) 5 × 10 nmols X X
I6279 Insulin (bovine) 5,730.6087 (monoisotopic) 5,734.51 (average)
5 × 10 nmols X X
C8857 Cytochrome c (equine) 12,361.96 (average) 5 × 10 nmols X X
A8971 Apomyoglobin (equine) 16,952.27 (average) 5 × 10 nmols X X
A9096 Aldolase (rabbit muscle) 39.212.28 (average) 5 × 10 nmols X X
A8471 Albumin (bovine serum) 66,430.09 (average) 5 × 10 nmols X X
Components
Calibration Mix, Standard Range 5 vials, each containing MRFA, Bradykinin 1–7, Angiotensin 1, Neurotensin, Renin Substrate, and Bradykinin
Calibration Mix, High Range 5 vials, each containing MRFA, Bradykinin, ACTH 1–16, Melittin, and ACTH 7-38
Sensitivity Standard Angiotensin II
Matrix α-cyano, 2 × 10 mg
Solvents 1% TFA Acetonitrile Ethanol, 200 Proof, Molecular Biology Grade
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157Our Innovation, Your Research — Shaping the Future of Life Science
Mass Spectrometry – Additional Reagents
Matrices for MALDI-MS Cat. No. Product Description Acronym Recommended Conditions Quantity
C8982 ProteoMass α-Cyano-4-hydroxycinnamic acid CHCA, ACCA Mass <10 kDa 10 × 10 mg
S8313 ProteoMass Sinapinic Acid, SA Mass >10 kDa 10 × 10 mg 3,5-Dimethoxy-4-hydroxycinnamic acid
54796 2-(4-Hydroxyphenylazo) benzoic acid HABA 1 g 5 g
85707 2,5-Dihydroxybenzoic acid DHB 250 mg 1 g
14078 Succinic acid IR-Laser 1 g 5 g
37468 2,6-Dihydroxyacetophenone UV-Laser 1 g 5 g
46278 Ferulic acid UV-Laser 1 g 5 g
60018 Caffeic acid UV-Laser 1 g 5 g
49771 Glycerol Liquid Matrix 1 ml 5 ml
72681 4-Nitroaniline Liquid Matrix 250 mg 1 g
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Isotec® Stable Isotopes for Biomolecular NMR
158 To place an order call your local office or visit sigma.com/order.sigma.com/proteomics
Products for Biomolecular NMRProduction of isotope labeled proteins has become easily accessible to today’s biochemists. A variety of isotope labeled substrates are readily available for feeding the genetically modified microorganisms, which produce the proteins of research interest. These growth media components include 13C-labeled glucose; 13C-; 15N-; 13C, D-, 15N, D-labeled amino acids; 15N-ammonium chloride; and even isotope-enriched growth media tailored for specific microorganisms. Alternatively, the proteins can be prepared from isotope labeled amino acids using cell free synthesis. As a result, scientists can harvest isotope labeled proteins and other interesting biomolecules for scientific research in a relatively short period of time, a process that was only imagined not long ago.
Minimal Media
Cat. No. Product Description Atom % Size
389374 d-Glucose-13C6 99% 13C 100 mg, 250 mg, 1 g, 10 g
552003 d-Glucose-C-d7 97-99% D 100 mg, 1 g, 10 g
552151 d-Glucose-13C6,C-d7 99% 13C, 97-99% D 500 mg, 1 g, 5 g
299251 Ammonium-15N chloride 98% 15N 250 mg, 1 g, 20 g
299286 Ammonium-15N2 sulfate 98% 15N 250 mg, 1 g, 10 g, 20 g
617385 Deuterium oxide 99.8% D 1 kg
Complex Growth Media
Cat. No. Product Description Atom % Size
606863 ISOGRO®-13C Powder -Growth Medium 99% 13C 1 g
606839 ISOGRO-13C,15N Powder -Growth Medium 99% 13C, 98% 15N 1 g
Alpha Keto Acids
Cat. No. Product Description Atom % Size
571334 2-Keto-3-(methyl-13C)-butyric acid-4-13C sodium salt 99% 13C 100 mg
607568 2-Keto-3-methylbutyric acid-13C5,3-d1 sodium salt 99% 13C, 98% D 250 mg
594903 2-Keto-3-(methyl-d3)-butyric acid-4-13C sodium salt 99% 13C, 98% D 100 mg
637858 2-Keto-3-(methyl-d3)-butyric acid-1,2,3,4-13C4, 3-d1 99% 13C, 98% D 250 mg
Additional Products of Interest
Cat. No. Product Description Atom % Size
485616 Dodecylphosphorylcholine-d38 98% D 500 mg
489379 Ethylenediaminetetraacetic-d12 acid 98% D 1 g
451851 Sodium dodecyl sulfate-d25 98% D 100 mg, 500 mg
655392 Bis-tris-d19 98% D 500 mg, 1 g
In addition to the products listed above, Sigma has all twenty amino acids uniformly labeled with 15N or 13C, along with the widest offering of selectively labeled amino acids available. For a complete listing of stable isotopes for BioNMR, please visit sigma.com/isotec.
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159Our Innovation, Your Research — Shaping the Future of Life Science
X-Ray Crystallography
Crystallography Kits
Basic Crystallography KitThe Basic Crystallography Kit is a rapid, empirical, and efficient screening method for determining the solubility and optimal starting conditions for the crystallization of biological macromolecules. Based on known and published data for various proteins, the Basic Kit uses a sparse matrix method and a minimal amount of protein sample to explore a broad range of buffers, pH, and precipitants.
Ordering InformationCat. No. Product Description Quantity
82009 Basic Crystallography Kit 1 kit
Basic Extension KitThe Extension Kit substantially increases the screening range of the Basic Crystallography Kit by providing a broader range of organic solvents, PEGs, and additives (divalent ions and stabilizers).
Both the Basic and Basic Extension Kit are based on the “salting-out” principle: that increasing the concentration of the precipitating agent depletes the macro-molecules of bound water and leads to their precipitation and crystallization.
Ordering InformationCat. No. Product Description Quantity
70437 Basic Extension Kit 1 kit
Membrane Protein KitThe Basic Crystallization Kit for Membrane Proteins is a rapid, empirical, and efficient screening method to determine the best conditions for crystallization of hydrophobic macromolecules (primarily membrane proteins) in an empirical way. Based on known or published data for various proteins, broad ranges of buffers, pH, and precipitants in combination with a micelle-forming detergent are explored using the sparse matrix method with a minimal amount of protein.
The crystallization principle of the kit is based on the screening of the hydro-philic, charged parts of the macromolecules by the binding of the detergent to hydrophobic areas of the macromolecules and subsequent reduction of their solubility. Detergent mediated contacts then lead to precipitation or crystalliza-tion of the screening molecules.
Ordering InformationCat. No. Product Description Quantity
73513 Membrane Protein Kit 1 kit
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X-Ray Crystallography
160 To place an order call your local office or visit sigma.com/order.sigma.com/proteomics
Cryo KitThe Crystallization Cryo Kit is a rapid, empirical, and efficient screening method to determine the solubility and optimal starting conditions for the crystallization of biological macromolecules in the presence of a cryoprotectant (primarily glycerol). Based on known or published data for various proteins, the Cryo Kit uses a sparse matrix method and minimal amount of protein sample to explore a broad range of buffers, pH, and precipitants. The screening range and probing ability of the Cryo Kit are comparable to those of the Basic Kit. A cryoprotectant is added to freeze crystals in an amorphous glass at –173 °C.
The Cryo Kit is also based on the “salting-out” principle: that increasing the concentration of precipitating agent and cryoprotectant depletes the macro-molecules of bound water and leads to their precipitation or crystallization.
Ordering InformationCat. No. Product Description Quantity
75403 Cryo Kit 1 kit
Low Ionic KitThe Crystallization Low Ionic Kit for proteins is based on a screening protocol for monoclonal antibodies, which has been proven to be an effective screen-ing method for soluble proteins. The high-efficiency of this kit can be further improved by pre-determining the isoelectric point (pl) of the subject macro-molecule followed by screening within a range at or near that value (within 2-3 pH units of the pl).
The Low Ionic Kit is based on the “salting-in” principle: that decreasing ion concentrations leads to a loss of electrostatic screening of charges on the macromolecules by low molecular weight ions. This loss of screening is compensated by the mutual screening of opposite charges on neighbor-ing macromolecules. This leads to decreased macromolecular solubility and subsequent precipitation or crystallization.
Ordering InformationCat. No. Product Description Quantity
86684 Low Ionic Kit 1 kit
For Crystallography Kit Components, visit sigma.com
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161Our Innovation, Your Research — Shaping the Future of Life Science
PEG Grid Screening KitThe PEG Grid Screening Kit is a rapid, systematic, and efficient screening method for determining the solubility and optimal starting conditions for the crystalliza-tion of biological macromolecules in the presence of high purity polyethylene glycol. It includes a series of crystallization conditions based on PEG 600 and PEG 6000 combined with a variety of high purity salts in comparably low con-centrations. It also displays a screen for pH 4 to 9. PEG is the most frequently used precipitant in crystallization; whereas, pH and ion strength are the main other variables to modify conditions.
Features and Benefitsn Comprehensive, efficient screening
n Extremely pure, sterile reagents
n Prefabricated reagents and observation sheets
n Low incidence of undesirable solubility effects
Ordering InformationCat. No. Product Description Quantity
36436 PEG Grid Screening Kit 1 kit
Crystallization Kit for Proteins for Automatic Screening96 Components of 1 ml each in a 8 × 12 deep-well plate. Kit covers most of the solutions contained in the Crystallization Basic Kit (82009) and Crystallization Extension Kit (70437).
Ordering InformationCat. No. Product Description Quantity
56783 Crystallization Kit for Proteins for 1 kit Automatic Screening
Find!
Our Innovation, Your Research — Shaping the Future of Life SciencePEPscreen® is a registered trademark of Sigma-Genosys, L.P.
Seek and find your peptide-binding region quickly with PEPscreen® Custom Peptide Libraries
Sigma-Genosys’ proprietary high-throughput peptide synthesis platform enables the quickest and most efficient production of peptide libraries at the milligram scale.
n Fast — delivery in 7 business days allowing for faster target identification
n Quality — peptide integrity confirmed via MALDI-TOF MS for each peptide
n Quantity — gross weight amount provided for each peptide
n Epitope Mapping
n Protein-Protein Interaction
n Peptide Microarray
n Screening Assays
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