Mass Spectrometry Sample Prep

Proper Mass Spectrometry (MS) sample preparation is crucial for obtaining high-quality MS data. Since samples originate from various biological sources, they can differ in protein concentration, buffer composition, and contaminants. We have refined our MS sample preparation methods to maximize peptide yield and minimize interference, leading to reliable downstream data interpretation. Please follow these sample submission guidelines when sending MS samples.

Typical MS Sample Preparation Workflow

  • Buffer Exchange: Protein samples are buffer-exchanged using spin columns into an SDS-based sample buffer.
  • Measure Protein Concentration: Protein concentration is measured and adjusted to the same across samples to ensure reliable MS comparisons.
  • Reduction & Alkylation: Disulfide bonds are reduced with dithiothreitol (DTT) followed by alkylation with iodoacetamide.
  • Protease Digestion: Proteins are digested with MS grade Trypsin & Lys-C or other proteases.

Common Challenges and Solutions for MS Sample Preparation: We offer the following solutions to address main challenges in different sample types. Customers are recommended to address these issues or notify us prior to sending samples. These additional steps in MS sample prep, such as protein concentrating, removing BSA/IgG and enrichment will lead to much better detection of low-abundance proteins.

Sample TypeMain ChallengeABO Customized Services
Culture media secreted proteinsLow protein concentration
May contain BSA		Protein concentrating
Remove BSA
Serum free media samplesContains serum proteinsRemove serum proteins
IP w/ non-crosslinked beadsHigh IgG contentRemove IgG
Cells / Tissues / HCPsHigh complexityFractionation by Mw and/or pI
Phospho proteomics samplesVery low phospho-peptide contentPhospho protein enrichment
Phospho peptide enrichment
Harvest with high HCP & DSHigh DS contentRemove DS

The following example shows the number of high confidence hits (containing >=2 peptides) in a sample containing BSA. After removing BSA by >90%, the total number of high confidence hits increased from 5 to 85.

Sample ConditionProteins Detected
>5 peptides>4 peptides>3 peptides>2 peptides
Before BSA removal1235
After BSA Removal20253685

Guidelines and Preparation for Common Sample Types

Please follow the table below for sample requirements, and our strategies for MS sample preparations. By following these optimized protocols, we ensure high reproducibility, minimal sample loss, and improved sensitivity in MS-based proteomics experiments. Customers may also view our general guidelines for sending samples.

Sample Type

Sample Requirements

ABO Sample Preparation

Gel Bands

  • Visible with Silver Staining or Coomassie.
  • Load a higher protein amount per lane instead of sending multiple gel pieces. Minimize gel volume when excising bands.
  • If using silver staining, must be formaldehyde-free to prevent crosslinking.
  • Gel cleaning to remove Dyes and MS interfering chemicals
  • In-gel reduction, alkylation and trypsin digestion

Immuno-precipitation (IP)

  • IP beads preferred
  • IP elutes: Please provide buffer composition
  • Proteins bound to IP beads are eluted
  • Perform buffer exchange to remove detergents and salts that interfere with MS

Tissue

  • Avoid any residual buffer
  • Flash-freeze samples in liquid nitrogen and store at -80°C; ship on dry ice.
  • Tissue is homogenized in lysis buffer, then exchanged into LCMSMS buffer.
  • Sonication or mechanical homogenization may be needed for tough tissues

Powder

  • Must be soluble.
  • Minimize/avoid contaminants that interfere with MS, such as salts, detergents, and polymers.
  • Powder is solubilized in a proper buffer followed by protein extraction, then exchanged into LCMSMS buffer

FAQ

Frequently asked questions about Mass Spec features, process, time & cost:

Our Protein ID service offers almost 100% success rate.

There are many factors that may affect a successful identification. The main factors are: low protein amount; proteins with significant amount of PTMs or chemical modifications, very hydrophobic proteins and proteins with few tryptic sites.

In nanoLC-MS/MS, digested peptides are first separated in a nano-flow LC, then subject to high-resolution orbitrap mass spectrometry. We have optimized protocols to identify all proteins in a protein mixture ranging from 1D gel bands to cell lysates.

Label-free quantitation uses the number of spectra or the intensity of spectra to compare the relative abundance between samples. Due to the high sensitivity of Orbitrap Mass Spectrometry and sophisticated Proteome Discoverer software, this is a reliable approach that is more straightforward than labeled methods.

TMT and iTRAQ use isobaric tags to label all peptides in a sample. Although the tags have the same total mass, the reporter regions have different masses. These differences are detected by the mass spectrometer after the fragmentation step in MS/MS, allowing highly accurate and precise quantitation.

The DDA approach fragments only peptide precursors meeting certain criteria, and searches against a protein database.

The DIA approach fragments all peptides across a precursor window, and searches the precursors against a spectral library database.

Although DIA produces more hits, it can generate more false positives than DDA. In addition, the time it takes software to process DIA data is significantly longer than for DDA.

Our LC-MS platform has a max resolution of 240,000 FWHM and dynamic range of 5 orders, allowing even the lowest abundance proteins to be detected.

5-7 days.