Proteome Mapping Methods
Proteome Mapping
To profile the content of a given protein sample, we employ an SDS-PAGE separation in the first dimension and then excise the lane into segments (the greater the number of segments, the deeper the coverage of the sample); each segment is then in-gel digested with trypsin and analyzed by nano LC/MS/MS. The number of proteins detected also increases with the amount of mass spectrometry time spent on the analysis. Each segment is searched against a concatenated forward and reverse database and the search results assembled into a non-redundant list from the entire lane using Scaffold. Through careful optimization of protocols at each stage of the process – SDS-PAGE, digestion, nano LC, mass spectrometry and database searching, NextGen is able to achieve outstanding levels of sensitivity and coverage. Here we show data for whole cell lysates, biofluids and sectretome samples.
Whole Cell
Example data is shown below for a THP-1 cell lysate (20µg loaded on the gel). The lane was excised into 40 segments and each digest analyzed on a 1h LC/MS/MS gradient with an LTQ Orbitrap XL mass spectrometer:
| THP-1 | |
| No. of Proteins (≥2 peptides/protein) | 3409 |
| No. of Unique Peptides | 32186 |
| Total Spectral Count | 56499 |
| Mean Unique Peptides/Protein | 9.4 |
| Mean Spectral Count/protein | 16.6 |
| Median Unique Peptides/Protein | 6 |
| Median Spectral Count/Protein | 8 |
| No. of Reverse Hits | 0 |
| Protein False Discovery Rate (%) | 0 |
| No. of Proteins (≥2 peptides/protein) | 3409 |
| No. of Proteins (≥3 peptides/protein) | 3001 |
| No. of Proteins (≥4 peptides/protein) | 2652 |
Bio Fluids
Example data is shown below for three biofluids: CSF, urine and plasma. The lanes were excised into 40 segments each and digests analyzed on a 1h LC/MS/MS gradient with an LTQ Orbitrap XL mass spectrometer:
| CSF | Urine | Plasma | |
| No. of Proteins (≥2 peptides/protein) | 850 | 894 | 440 |
| No. of Unique Peptides | 9175 | 8654 | 5839 |
| Total Spectral Count | 50359 | 39747 | 45678 |
| Average Unique Peptides/Protein | 10.8 | 9.7 | 13.3 |
| Average Spectral Count/protein | 59.2 | 44.4 | 103.1 |
| Median Peptides/Protein | 6 | 6 | 6 |
| Median Spectral Count/Protein | 14 | 12 | 10 |
| Protein False Discovery Rate (%) | <0.1 | <0.1 | <0.9 |
| No. of Proteins (≥2 peptides/protein) | 850 | 894 | 440 |
| No. of Proteins (≥3 peptides/protein) | 717 | 749 | 315 |
| No. of Proteins (≥4 peptides/protein) | 610 | 627 | 278 |
Secreted Proteins
Example data is shown below for secreted proteins isolated from spent media of cultured A549 cells. Approximately 7 million cells were grown and half the spent media loaded on the gel (~20µg). The lanes were excised into 40 segments each and digests analyzed on a 1h LC/MS/MS gradient with an LTQ Orbitrap XL mass spectrometer:
| A549 Secretome | |
| Total no. of proteins | 1455 |
| Total Spectral Count | 48092 |
| Total Unique Peptides | 15148 |
| Mean Spectral Count | 33.1 |
| Median Spectral Count | 10 |
| Mean Unique Peptides | 10.4 |
| Median Unique Peptides | 6 |
| No. of reverse hits | 0 |
| % FDR | 0 |