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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  
Fast  protein  digestion  for  Peptide  Mass  Fingerprinting  
 
 
 
 
+
Protocol  
Fast  protein  digestion  for  Peptide  Mass  Fingerprinting
 
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.  
 
 

 
ProteoGen  Bio   3  
Fast  protein  digestion  for  Peptide  Mass  Fingerprinting  
 
 
 
 
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  
Equilibration  buffer   Digestion  buffers  
40  mM  ammonium  bicarbonate,  pH  7.8   40  mM  ammonium  bicarbonate,  pH  7.8  
 
Buffers  suggested  for  DigesTip  Pepsin  
Equilibration  buffer   Digestion  buffers  
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  
Fast  protein  digestion  for  Peptide  Mass  Fingerprinting  
 
 
 
 
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  
Fast  protein  digestion  for  Peptide  Mass  Fingerprinting  
 
 
 
 
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  

 
ProteoGen  Bio   6  
Fast  protein  digestion  for  Peptide  Mass  Fingerprinting  
 
 
 
 
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.  

 
ProteoGen  Bio   7  
Fast  protein  digestion  for  Peptide  Mass  Fingerprinting  
 
 
 
 
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).  

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

 
ProteoGen  Bio   8  
Fast  protein  digestion  for  Peptide  Mass  Fingerprinting  
 
 
 
 
 

 
 
 

ProteoGen  Bio  Srl  


Viale  Rinaldo  Piaggio  32,  56025,  Pontedera  (PI),  Italy  
Phone  +39  0587-­‐274816  -­‐  Fax  +39  0587  970085    
Web  www.proteogenbio.com  
*  suggested  for  DigesTip  10  μl  format  
**  suggested  for  DigesTip  100  μl  format