Fast Protein Digestion For Peptide Mass Fingerprinting

Fast protein digestion

for Peptide Mass
Fingerprinting

ProteoGen Bio 1
Fast protein digestion for Peptide Mass Fingerprinting

Summary

Introduction ............................................................................................................................... 2
Equipment .................................................................................................................................. 3
Reagents ..................................................................................................................................... 3
Sample preparation .................................................................................................................... 4
DigesTip preparation .................................................................................................................. 5
Digestion .................................................................................................................................... 5
Processing of digested sample .................................................................................................... 5
Mass Spectrometry analysis ........................................................................................................ 6
Troubleshooting ......................................................................................................................... 7

ProteoGen Bio 2

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Protocol

suitable for
DigesTip Trypsin, DigesTip Chymotrypsin, DigesTip Glu-‐C and DigesTip Pepsin
The easiest and fastest way to digest proteins
10 μl (100 μl) pipette tip-‐shaped bioreactor for sample digestion

Introduction

Proteomics, the study of all proteins expressed by a genome, has become in recent decades an important science of
the post-‐genomic era. A significant focus in proteomic studies is the identification of proteins. Proteins digestion and
their separation are two critical steps for analysis and identification of proteins by Mass Spectrometry. Furthermore, a
reproducible cleavage pattern of digested proteins is a prerequisite for their unambiguous identification by Mass
Spectrometry.

Peptide Mass Fingerprinting (PMF) is an analytical technique that allows identifying proteins by cleaving them into
peptides, whose masses are then accurately measured by Mass Spectrometry and used to query a database.
Traditional digestions are performed in solution by proteases. This step however leads to different issues such as
extended incubation times, low efficiency and enzyme auto-‐digestion.

ProteoGen Bio has developed an innovative method for modification of active proteins and their immobilization on a
solid surface, thus generating monolayers of oriented molecules that fully preserve their activity. DigesTip is a novel
tip-‐shaped device for protein digestion, containing a filter with active immobilized proteases, usable both manually
and by robots. Its key features are a very short digestion time, high efficiency in a wide range of protein sample
concentrations, and the ability to provide a peptide mixture readily suitable for Mass Spectrometry measurements or
other applications.
This protocol describes the use of DigesTip Trypsin, DigesTip Chymotrypsin, DigesTip Glu-‐C and DigesTip Pepsin for
digesting a protein sample and identifying proteins contained in it simply by using only one DigesTip. This new
approach allows a fast and efficient protein digestion, readily suitable for PMF.

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Equipment

A DigesTip selected from DigesTip Trypsin, DigesTip Chymotrypsin, DigesTip Glu-‐C or DigesTip Pepsin is required for
this protocol.
DigesTip requires a compatible single or multichannel pipettor.
Samples can be dispensed and digested in microcentrifuge tubes or in separated wells of a multiwell polypropylene
microplate.

In addition, the following optional equipment may be needed:
ü for protein reduction, an oven or a thermoblock capable to reach at least 60°C;
ü for protein separation, a liquid chromatography system (e.g. reverse-‐phase, ion-‐exchange or affinity methods);
ü for sample purification, a liquid chromatography system (e.g. gel-‐filtration or reverse-‐phase methods) or a dialysis
tubing;
ü for desalting of digested sample, a reverse-‐phase C18 pipette tip.

Reagents

For a proper use of DigesTip two distinct buffers are required: an equilibration buffer (for filter equilibration) and a
digestion buffer (for the digestion step). Buffers that guarantee the best results are shown below. If needed, buffers
different from those suggested can be used both for equilibration and digestion.

Buffers suggested for DigesTip Trypsin and DigesTip Chymotrypsin
Equilibration buffer Digestion buffers
40 mM ammonium bicarbonate, pH 8 40 mM ammonium bicarbonate, pH 8

Buffers suggested for DigesTip Glu-‐C
40 mM ammonium bicarbonate, pH 7.8 40 mM ammonium bicarbonate, pH 7.8

Buffers suggested for DigesTip Pepsin
100 mM sodium citrate, pH 2 100 mM sodium citrate, pH 2
0.5 M formic acid, pH 2 0.5 M formic acid, pH 2

For DigesTip Pepsin, it is possible to choose the buffer that best reflects your needs from those indicated in the table.
For Matrix-‐Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry applications, the presence of sodium
citrate should not affect the results, but for analysis with Electrospray Ionization (ESI) Mass Spectrometry and for
other applications, the presence of non-‐volatile salts may interfere with the analysis, i.e. suppressing the ionization. In
this case, 0.5 M formic acid, pH 2 is recommended, instead of sodium citrate buffer.

In addition, the following optional reagents may be needed:
ü for protein denaturation, a chaotropic agent like guanidine or urea;
ü for protein reduction, a reducing agent like dithiothreitol (DTT);
ü for protein alkylation, iodoacetamide or iodoacetic acid;
ü for protein deglycosylation or dephosphorylation, peptide N-‐glycosidase F (PNGase F) or alkaline phosphatase,
respectively.

ProteoGen Bio 4

Sample preparation

1. Separate your proteins by liquid chromatography
Protein separation represents a key factor for an efficient analysis and may influence PMF results. A biological sample
typically contains several proteins, and this complexity requires some separation steps in order to increase
the detection of low-‐abundance proteins. Liquid chromatography is the most widely used technique for protein
separation, according to their own unique characteristics. A single separation step may not be sufficient, so a multi-‐
dimensional separation may be needed, i.e. combining reverse-‐phase and ion-‐exchange chromatographies.
Alternatively, affinity chromatography can be used in some cases, e.g. in glycoprotein identification.

2. Deglycosylate or dephosphorylate your sample (optional)
Some proteins, such as glycoproteins or phosphoproteins, could require a further processing. For example, PNGase F
can be used to remove the carbohydrate groups attached to glycoproteins. Similarly, it could be useful to
dephosphorylate phosphoproteins with alkaline phosphatase. For a detailed protocol refer to the specific manual
available from your enzyme supplier.

3. Alkylate your protein (optional)
This step can be skipped because it is possible to digest also native proteins, without a previous reduction or
alkylation. In this case, sample does not require an additional purification and the step 4 can be skipped. However, a
complete digestion may require denaturation of the protein and reduction of its disulfide bridges. This step is
suggested to prevent cysteines from combining in disulfide linkages, in order to consider also cysteine-‐containing
peptides in the analysis. Moreover, alkylation makes the protein digestion easier and more efficient. Sample can be
reduced incubating it with 50 mM DTT at 60°C for at least 10 minutes, in 100 mM ammonium bicarbonate, pH 8,
containing guanidine 6 M or urea 8 M as chaotropic agent. Sample should be now alkylated incubating it with 100 mM
iodoacetamide or iodoacetic acid at Room Temperature for at least 30 minutes, in the dark.
Alternatively, it is possible to digest the sample without alkylating it, following the specific protocol available on
ProteoGen Bio website. This protocol allows reducing a protein sample in only 10 minutes, simply using DTT and
acetonitrile or an acid-‐labile surfactant. In this case, sample does not require an additional purification and the
step 4 can be skipped.

4. Purify the sample from contaminants (optional)
Depending on the composition of buffers used in the previous steps, sample may need to be purified from salts and
other contaminants (e.g. chaotropic agents) before digestion. For an updated list of compatible reagents refer to the
protocol section on ProteoGen Bio website.
Sample can be purified by dialysis, size-‐exclusion chromatography or reverse-‐phase chromatography. For the first
hypothesis, sample should be dialyzed against the digestion buffer. Purification by size-‐exclusion chromatography
TM
could be achieved using a GE Healthcare Sephadex G-‐25 column and eluting directly with the digestion buffer.
TM
Furthermore, purification by reverse-‐phase chromatography could be obtained using Millipore ZipTip or Agilent
TM
Omix C4 pipette tips. For detailed protocols refer to specific manuals available from GE Healthcare, Millipore or
Agilent.

ProteoGen Bio 5

5. Prepare an aliquot of the sample
Prepare an aliquot of 2 μl* (50 μl**) of the protein sample by dissolving or diluting it with the digestion buffer. For this
protocol, DigesTip Trypsin and DigesTip Chymotrypsin work optimally with protein concentrations from 5 μg/ml to 2
mg/ml, DigesTip Glu-‐C with protein concentrations from 20 μg/ml to 1 mg/ml, and DigesTip Pepsin with protein
concentrations from 100 μg/ml to 500 μg/ml.
Bring the sample to Room Temperature prior to digest it with DigesTip.

DigesTip preparation

6. Plug a DigesTip into the pipettor
Remove the DigesTip from the package. Before starting the digestion procedure, make sure that the DigesTip is at
Room Temperature.
The filter at the tip of DigesTip provides a slight back pressure. Therefore, it is recommended to set the pipettor to 10
μl* (100 μl**) or more. Plug the DigesTip into the pipettor. Press the pipettor plunger to the first stop and hold it in
this position.

7. Equilibrate the DigesTip
Dip the DigesTip into 50 μl* (500 μl**) of equilibration buffer and pipette the sample, by pressing and releasing for 5
times the plunger, in order to ensure the complete wetting of the resin.
Empty the DigesTip of the excess buffer. Make sure that the DigesTip does not dry before the digestion.

Digestion

8. Digest your sample with the DigesTip
Keep the equilibrated DigesTip plugged to the pipettor.
Dip the DigesTip into 2 μl* (50 μl**) of the previously prepared protein sample and pipette the sample, by pressing
and releasing repeatedly the plunger for at least 1 minute, to ensure the digestion of the protein sample. For DigesTip
Glu-‐C and DigesTip Pepsin, a digestion time of 2 minutes is recommended. For DigesTip Trypsin and DigesTip
Chymotrypsin, instead, a digestion time of 1 minute should be enough. However, a longer digestion time can be used
for hard-‐to-‐digest proteins. Make sure the DigesTip remains immersed in the solution and the entire filter is wet with
the sample while pipetting in order to avoid the formation of air bubbles.

9. Recover your digested sample
Press the plunger completely to recover the entire digested sample, now ready to be analyzed or further processed.

Processing of digested sample

10. Purify the digested sample from salts or other contaminants (optional)
Depending on the Mass Spectrometer used, it could be useful to purify the digested sample from unwanted salts or

* suggested for DigesTip 10 μl format
** suggested for DigesTip 100 μl format

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TM
other contaminants before the analysis, for example using a reverse-‐phase C18 pipette tip like Millipore ZipTip or
TM
Agilent Omix , able to bind, concentrate and elute digested peptides. For detailed protocols refer to specific manuals
available from Millipore or Agilent.

11. Reduce the sample (optional)
If the sample has been digested without a previous reduction or alkylation, it should be reduced prior to analyse the
digest, using 50 mM DTT at 60°C for at least 10 minutes.

Mass Spectrometry analysis

12. Analysis of the digested sample by Mass Spectrometry
Either a matrix-‐assisted laser-‐desorption ionization (MALDI) or an electrospray ionization (ESI) technique could be
chosen for analysis. MALDI is a solid-‐state technique in which a mixture of sample and matrix is spotted on a target
plate. ESI, in contrast, is a flow-‐based technique in which sample droplets, often coming from a chromatographic
system, are ionized by an electrically charged nozzle. An advantage of MALDI is that it is faster than ESI and enables
higher throughput, but ESI is more sensitive. The choice depends on the nature of the sample and the information
needed.

13. Setting of the correct parameters before querying the database
Before querying the database with peak list, the missed cleavages should be set to 3 for DigesTip Trypsin, 7 for
DigesTip Chymotrypsin, 3 for DigesTip Glu-‐C and 7 for DigesTip Pepsin.
If the sample has been alkylated before digestion, the correct fixed modification should be considered in the query. It
could be useful to consider also some variable modifications like methionine oxidation or serine and threonine
phosphorylation. If a glycosidase like PNGase F has been used to remove carbohydrate groups, asparagine has been
converted into aspartic acid and asparagine deamidation should be chosen as variable modification.
Be sure that the correct cleavage sites are considered for querying the database. For DigesTip Trypsin, set the
carboxy-‐side of lysine and arginine as cleavage sites. For DigesTip Glu-‐C, set the carboxy-‐side of glutamic acid. The
carboxy-‐side of tyrosine, phenylalanine, tryptophan, leucine and methionine should be considered as cleavage sites
for DigesTip Chymotrypsin. For DigesTip Pepsin, set the carboxy-‐side of phenylalanine, leucine, alanine and glutamic
acid.

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Troubleshooting

The following table outlines common problems and their possible causes, and suggests procedures that could be
helpful to solve these troubles.

The mass spectrum shows few or no peaks
Possible Cause Suggested Procedure

One or more protocol steps were not completed Check that the correct reagents and conditions were used.
properly.
The protein concentration is too low. Increase the number of laser “shots” during the spectrum acquisition
process.
The protein is difficult to digest. Extend the digestion time. With the same DigesTip keep on digesting the
protein sample by pipetting for a longer time. It could be helpful to follow
the suggestions reported in step 3 of the “Sample Preparation” section.
Protein loss may have occurred during storage. Use low-‐binding microtubes for sample storage.
Some peptides could have been retained into the Elute peptides from the filter by pipetting 2 μl* (50 μl**) of a 50%
digesting filter. acetonitrile (ACN) solution with the same DigesTip used for digestion.
The matrix to analyte ratio on the spot is too high The matrix concentration (e.g. cyano-‐4-‐hydroxycinnamic acid) has to be
(MALDI). reduced.

Peptide peaks were picked, but no protein was identified
Possible Cause Suggested Procedure

Protein has been modified before digestion. Set the correct protein modification in the fixed modification setting
before querying the database (see step 13 of “Mass Spectrometry
analysis” section).
Digested proteins could be variably modified (e.g. Set the appropriate variable modification before querying the database
phosphorylated). (e.g. phosphorylation of serine and threonine). Deamidation or
methionine oxidation can also occur in some circumstances and should be
considered (see step 13 of “Mass Spectrometry analysis” section).
Sample is too complex. Digests of complex protein mixtures are not suited for PMF. For this
reason, some separation steps are necessary for reducing the complexity
of the sample prior to digestion.
Too few cleavage sites are considered. For DigesTip Chymotrypsin, set the cleavage sites including carboxy-‐side
of methionine in addition to tyrosine, phenylalanine, tryptophan and
leucine before querying the database. For DigesTip Pepsin, set the
cleavage sites including carboxy-‐side of alanine and glutamic acid in
addition to leucine and phenylalanine before querying the database.
Missed cleavage setting is too restrictive. Set the missed cleavage setting to 3 for DigesTip Trypsin, 7 for DigesTip
Chymotrypsin, 3 for DigesTip Glu-‐C and 7 for DigesTip Pepsin, before
querying the database.
The spectrum is not well calibrated. Review the calibration procedure or try less restrictive tolerance settings
to query the database.
The protein is unknown (sequence not present into None.
the database).


ProteoGen Bio 8

ProteoGen Bio Srl

Viale Rinaldo Piaggio 32, 56025, Pontedera (PI), Italy
Phone +39 0587-‐274816 -‐ Fax +39 0587 970085
Web www.proteogenbio.com

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Fast protein digestion

4. Purify the sample from contaminants (optional)

Keep the equilibrated DigesTip plugged to the pipettor.

Possible Cause Suggested Procedure

Possible Cause Suggested Procedure

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