Services Described

Molecular weight of an intact protein determined by MALDI MS

This method has low femtomol sensitivity

 MALDI spectrum

                                                                          

Molecular weight of a small molecule determined by electrospray MS

MS small molecule

HPLC separation of small molecules

HPLC

                                                                             retention time, min

1D gel electrophoresis

Native or denaturing conditions, pictured gel had 10 or 20 ug of yeast lysate per lane

1D gel 

2D gel electrophoresis

  • separation by isoelectric point and molecular weight

  • A 11 cm gel requires 50 ug of protein

    2D gel silver

Stains available

Stain Protein detected Sensitivity
Coomassie blue total 20-50 ng
Silver total 5 ng
SYPRO Ruby total 1 ng
Pro Q Diamond phosphoproteins  1-16 ng
Pro Q Emerald glycoproteins  0.5 ng

  


Protein identification by liquid chromatography tandem mass spectrometry (LC-MS/MS)

  • Digestion of protein in solution or in gel with appropriate protease

  • Peptides fractionated by nano HPLC then introduced directly into a mass spectrometer

  • Data dependent MS/MS (selection and fragmentation of peptide ions)

  • Database searching for peptide identification

    • Peptides identified using Sequest, X!Tandem, and MASCOT algorithms

    • Protein identifications presented and analyzed using SCAFFOLD

    • Secure on-line results at our work request website: www.protbase.org

Our instruments can detect peptides in low femtomole concentrations (instruments are tested with 50 fmol of trypsin-digested bovine serum albumin).

 MS/MS peptides

Scaffold output

Scaffold output

Analysis of a Complex Protein Mixture

Analysis of a complex mixture can be done several ways. The simplest is a solution digest followed by one LC MS/MS run. The number of protein identifications depends mainly on the relative abundances of the sample's proteins. Proteins in low abundance (often the most interesting ones) are difficult to detect because the signals can be overwhelmed by the abundant proteins.

Fractionation reduces the complexity and, in general, yields far more protein identifications. Currently we have three methods for fractionation: analysis of regions from a gel lane, SCX of tryptic peptides off line, or SCX of tryptic peptides on line (MudPIT, described below). We have compared gel-based versus off-line SCX of a yeast lysate, see the following graph. The gel lanes contained 10 ug of protein, the SCX fractionation used 55 ug. As demonstrated below, SCX fractionation gave significantly more identifications, but, depending on the sample, and/or the researchers needs, a gel-based analysis may be more suitable. For example, if the sample is available in very limited quantities or if there are MS interfering components (such as CHAPS) that are not removed by the steps involved in the SCX fractionation.

SCX vs Gel 

 MudPIT

  • Multidimensional Protein Identification Technology, MudPIT, (or LC LC MS/MS), developed by the Yates research group at the Scripps Research Institute

  • Uses two in-line chromatography columns, typically strong cation exchange (SCX) and reverse phase (C18), to separate complex mixtures of proteins before introduction into the MS

  • Sample binds to the SCX and peptides are eluted off with increasing salt concentrations (salt bump)

  • SCX fractions flow directly onto the C18 column where the peptides are separated based on hydrophobicity

  • C18 separated peptides are nano sprayed into a mass spectrometer where their masses (MS) and fragments (MS/MS) are measured

  • Total cell lysate can be analyzed by this technique and the less abundant proteins will be detected as well as the abundant proteins

  • We need 10-30 ug total protein per MudPIT, if precise protein concentrations are required, the samples will be assayed by the Facility prior to protease treatment

  •  Experiments should be done in triplicate to maximize the number of proteins detected

 MudPIT

Protein purification

Methods can include:

  • Chromatography (ion exchange, hydrophobic interaction, affinity, gel filtration)

  • ÄKTA explorer FPLC

  • HPLC

  • Preparative isoelectric focusing

Removal of 12 abundant proteins from biological samples

  • Immunoaffinity chromatography using IgY antibodies

  • Removes 12-highly abundant proteins (albumin, IgG, transferrin, fibrinogen, α1-antitrypsin, IgA, IgM, α2-macroglobulin, haptoglobin, apolipoproteins A-I and A-II, and orosomucoid (α1-acid glycoprotein))

  • Less abundant proteins may then be detected to investigate the more clinically relevant sub-proteome

 IgY run

Immunoaffinity chromatography of 50 ul human serum using IgY LC12 column and FPLC. Bound proteins were eluted with 100 mM glycine pH 2.5.

Sample concentration and clean up

  • Methods are sample dependent

  • Can include dialysis, ultrafiltration, solid phase extraction, precipitation

 Protein Assays

  • Protein detection limits with our current microplate reader filters: Pierce 660nm: 250 ug/ml, , Bradford: 125 ug/ml, BCA: 125 ug/ml, micro BCA: 25 ug/ml