| Advanced MS Sample Prep and In-Gel Digestion | |
Using Millipore's unique microvolume chromatography technology
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- Concentration and desalting in seconds
- In-gel digestion and MS sample prep in one device
- Manual and automated solutions
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Millipore's unique "Zip" chromatography devices are the fastest and easiest way to digest and/or purify proteins, peptides, or oligonucleotides prior to mass spectrometry..
ZipPlate(tm) Micro-SPE Plates- Integrated reaction chamber and C18 cleanup eliminates steps, minimizes peptide loss, and improves sensitivity
- C18 micro-chromatography bed provides predictable scale-up from ZipTip and allows elution in MS-compatible buffer
- Standard 96-well format is compatible with all liquid handling systems
ZipPlate is a 96-well microtiter plate with 300 nanoliters of C18 media immobilized at the bottom of each well.
It is the first device of its kind to integrate digestion, desalting, and sample concentration into one device. Simply place the gel slices into the wells of the plate; digest the samples; and elute the digested peptides into a receiver plate.
Montage® In-Gel DigestZP Kit- Includes ZipPlate for one-plate in-gel digestion and sample preparation without transfer steps that can reduce sensitivity
- Prepared, quality-controlled reagents and an optimized protocol ensure the highest reliability
This new and improved Montage kit is the fastest and most efficient way to go from gel slice to purified, concentrated sample.
Prepared, quality-controlled reagents save time and minimize the variability inherent in in-house reagent formulation. In addition, the vacuum-based protocol eliminates tedious pipetting and sample transfers. Automated protocols are available.
The kit includes Promega Modified Trypsin, the laboratory standard for protein digestion.
ZipTip® Pipette Tips- The standard for microvolume MS sample preparation
- Available with 4 different chromatography media
- Compatible with pipettors, HPLC needles, or automated liquid handlers
ZipTips are ideal for low/mid-throughput research applications. The 10 µL pipette tips are available with a variety of chromatography media immobilized at their ends.
Samples are bound by aspirating through the resin several times. Contaminants are washed away and concentrated, purified sample can be eluted directly onto a MALDI target in 1 - 4 µL of MS-compatible buffer.
ZipTipµ-C18 contains a micro-bed of resin for elution volumes as low as 0.5 µL .
ZipPlate and Montage In-Gel DigestZP Kit
Performance
Simplified Protocol Saves Time and Improves Sensitivity
Figure 1. Performing in-gel digests in a micro-centrifuge tube and cleaning-up the digest with ZipTipµ-C18 is the current gold standard in the protein chemistry laboratory. ZipPlate simplifies the protocol by eliminating the need for a vacuum centrifuge step. As the data show, ZipPlate also provides superior sensitivity as measured by sequence coverage.
Effective Contaminant Removal
| Contaminant | Direct Spotting onto
a MALDI Target | Sample Clean-Up
with ZipPlate |
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| 8M Guanidine | | Score: 44
Peptides matched: 15 |
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| 1M NaCl | Score: 23
Peptides matched: 9 | Score: 120
Peptides matched: 21 |
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| 8M Urea | | Score: 48
Peptides matched: 13 |
Table 1. Peptide concentration and desalting is the most common ZipPlate application. To demonstrate desalting effectiveness, guanidine, NaCl, and urea were spiked into a 200 fmol beta-galactosidase tryptic digest. The table summarizes the efficiency of contaminant removal as demonstrated by Mascot® scores and peptides matched in the Swiss-Prot® database.
Improved Data Quality for Low Abundance Proteins
Figure 2. To evaluate ZipPlate's ability to concentrate and purify peptides from in-gel digests of low abundance proteins,100 fmol BSA was loaded onto a gel and then excised and digested and concentrated in ZipPlate. The resulting spectrum (ABI Voyager® STR Biospectrometry® Workstation in reflector mode) shows high quality and high confidence in protein identification (9 of13 peptides matched and MOWSE score of 2498). Note: spectrum shown is not deisotoped.
Reproducible Results
Figures 3, 4. ZipPlate was evaluated for well-to-well reproducibility with in-gel and in-solution digests.
ZipPlate is a product of the Millipore/Applied Biosystems Proteomics Collaboration.
ZipTip Pipette Tips
Performance
Figure 5. Equine myoglobin digest in the presence of urea and salt. Direct spotting of the digest was compared to samples prepared with ZipTipµ-C18. Results show that the micro-bed ZipTips significantly improve the resolution of the MS results by removing salts and urea. Data obtained on a Micromass TofSpec-2E(tm) in reflectron mode using ß-cyano-4-hydroxycinnamic acid matrix. The digest was a 3 pmol/µL equine myoglobin trypsin digest containing 0.2 M urea,10 mM NH4HCO3, 1mMDTT, and 2 mM lodacetamide. Data courtesy of Dr. Thomas Neubert, NYU School of Medicine.
Figure 6. An 80 fmol BSA digest in 0.1% TFA/20% acetonitrile was concentrated and purified with ZipTipSCX. A database search of the resulting peptide map successfully identified the protein with 28% coverage. Data obtained on an ABI Voyager-DE Biospectrometry Workstation.
Increase MALDI-TOF MS Sensitivity and Resolution by Fractionating Complex Mixtures
Figure 7. Step gradient elution following in-gel digestion of human IgG heavy chain. In addition to concentrating and desalting, step gradient elution with increasing acetonitrile serves to fractionate complex peptide mixtures and simplify spectra. One ZipTipC18 pipette tip was used to bind peptides from an in-gel digest of IgG heavy chain. Salts were removed by washing before bound peptides were sequentially eluted in the presence of matrix with increasing acetonitrile concentration. The simplified spectra allowed complete ionization of biomolecules that were otherwise quenched by competing ions. This approach increased both sensitivity and resolution of the MALDI-TOF MS. Data obtained on a ABI Voyager-DE Biospectrometry Workstation.
Desalt Proteins From Cell Lysates
Figure 8. Protein mixture isolated from E. coli lysate. Sample buffer contained 20 µM NaH2PO4, 300 mM NaCl, and 100 mM imidazole. Direct spotting of 5 pmol of protein mixture was compared to sample prepared with ZipTipC4 pipette tip. Spectra following desalting and concentration with ZipTip pipette tip showed substantial signal improvement. Data acquired on a Bruker Daltonics BiFlex III(tm) MALDI-TOF MS. Sample courtesy of Dr. Alex Buko, Abbott Laboratories.
Recommended Equipment
ZipPlate/Montage In-Gel DigestZP Kit
ZipPlate devices provide high throughput, parallel sample processing with both manually-operated multichannel pipettors or automated liquid handlers. Contact Millipore for available automated protocols.
ZipTip Pipette Tips
Manual Liquid Handling
Recommended for use with most single-channel pipettors. The VistaLab ULTRON(tm) Handheld Liquid Handling System with ZipTip Adapter Kit (Vista Lab part number 1052-9016) is recommended as a variable speed, programmable electronic pipettor. The Biohit Proline® electronic multi-channel pipettor is recommended for simultaneously processing eight samples.
Automated Liquid Handling
ZipTip pipette tips are proven for use on:
ABI SymBiot(tm) Sample Workstations for Biospectrometry with Universal Cone for ZipTip (ABI part number GEN602987) and Pipette Tip Tray for ZipTip box (ABI part number GEN601860)
Genomic Solutions ProMS(tm) Sample Preparation Station Bruker Daltonics MAP(tm) II and MAP II/8 MALDI AutoPrep System with rack adapter for ZipTip box (Millipore part number ST0BDAPTR)
Micromass 2700-MS Sample Preparation Station and MassPREP(tm) Station
Packard BioSciences MultiPROBE® Automated Liquid Handling System
Tecan GENESIS Workstation
CyBio CyBi(tm)-Well 96 Automated Pipettor
Specifications
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| Format | | |
| Well volume (maximum) | | |
| Working volume | | |
| Bed volume | | Standard: 0.6 µL
Micro-bed: 0.2 µL |
| Gel plug compatibility | | |
| Volume to immerse gel plug | | |
| Elution volume | 15 µL (vacuum)
4 µL (centrifuge) | |
Recommended centrifuge
speed (elution) | 1750 x g (3000 rpm)
for 15 seconds | |
| Material | | |
| Resin | | Several choices
(see below) |
Selection Guide
ZipTip
| Application | |
| Desalting/concentrating peptides & small proteins | |
| Step-fractionation of complex peptide mixtures | |
| Desalting/concentrating proteins | |
| Removing zwiterionic and nonionic detergents | |
| Concentration of peptides from organic solutions | |
| Enriching phosphopeptides | |
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