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. 2008 Dec;7(12):5286-94.
doi: 10.1021/pr8004666.

Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry

Robbert J C Slebos  1 , Jonathan W C Brock, Nancy F Winters, Sarah R Stuart, Misti A Martinez, Ming Li, Mathew C Chambers, Lisa J Zimmerman, Amy J Ham, David L Tabb, Daniel C Liebler
Affiliations

Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry

Robbert J C Slebos et al. J Proteome Res. 2008 Dec.

Abstract

Shotgun proteome analysis platforms based on multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a powerful means to discover biomarker candidates in tissue specimens. Analysis platforms must balance sensitivity for peptide detection, reproducibility of detected peptide inventories and analytical throughput for protein amounts commonly present in tissue biospecimens (< 100 microg), such that platform stability is sufficient to detect modest changes in complex proteomes. We compared shotgun proteomics platforms by analyzing tryptic digests of whole cell and tissue proteomes using strong cation exchange (SCX) and isoelectric focusing (IEF) separations of peptides prior to LC-MS/MS analysis on a LTQ-Orbitrap hybrid instrument. IEF separations provided superior reproducibility and resolution for peptide fractionation from samples corresponding to both large (100 microg) and small (10 microg) protein inputs. SCX generated more peptide and protein identifications than did IEF with small (10 microg) samples, whereas the two platforms yielded similar numbers of identifications with large (100 microg) samples. In nine replicate analyses of tryptic peptides from 50 microg colon adenocarcinoma protein, overlap in protein detection by the two platforms was 77% of all proteins detected by both methods combined. IEF more quickly approached maximal detection, with 90% of IEF-detectable medium abundance proteins (those detected with a total of 3-4 peptides) detected within three replicate analyses. In contrast, the SCX platform required six replicates to detect 90% of SCX-detectable medium abundance proteins. High reproducibility and efficient resolution of IEF peptide separations make the IEF platform superior to the SCX platform for biomarker discovery via shotgun proteomic analyses of tissue specimens.

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Figures

Figure 1
Figure 1
Effect of ampholyte concentration on peptide and protein identifications from tryptic peptides corresponding to 100 µg RKO cell lysate. Peptides were fractionated with the indicated concentrations of ampholytes on a 24 cm IGPhor pI 3.5–4.5 strip and 10 fractions were collected and analyzed by LC-MS/MS on an LTQ-Orbitrap instrument. Identifications from all 10 fractions were combined and represented for each ampholyte concentration.
Figure 2
Figure 2
Fractionation of RKO cell tryptic peptides by IEF (A, B) and SCX (C, D). Digests corresponding to either 10 µg (A, C) or 100 µg (B, D) of protein were fractionated in triplicate on either a 24 cm IGPhor pI 3.5–4.5 strip or on a capillary SCX column with a step gradient as described under “Materials and Methods”. For both IEF and SCX, 10 fractions were collected and analyzed by LC-MS/MS on an LTQ-Orbitrap instrument.
Figure 3
Figure 3
Efficiency of resolution of RKO cell tryptic peptides by IEF (A, B) and SCX (C, D) in analyses depicted in Figure 1. Pie charts depict percentages of peptide identifications associated with only one fraction, two fractions, three fractions or more than three fractions.
Figure 4
Figure 4
Accumulation of peptide (A) and protein (B) identifications in triplicate analyses of RKO cell tryptic peptides by IEF or SCX. Aliquots of peptide mixtures corresponding to 10 or 100 µg of protein were fractionated in triplicate on either a 24 cm IGPhor pI 3.5–4.5 strip or on a capillary SCX column with a step gradient as described under “Materials and Methods”. For both IEF and SCX, 10 fractions were collected and analyzed by LC-MS/MS on an LTQ-Orbitrap instrument. Bar graph shading indicates peptides and proteins found in only a single replicate analysis, in two of three replicates and in all three replicates.
Figure 5
Figure 5
Accumulation of protein group identifications during nine successive replicate analyses of tryptic peptides from colon adenocarcinoma tissue. Each replicate analysis corresponding to 50 µg of tissue protein. Peptides were fractionated on either a 24 cm IGPhor pI 3.5–4.5 strip or on a capillary SCX column with a step gradient as described under “Materials and Methods”. For each replicate analysis by both IEF and SCX, 10 fractions were collected and analyzed by LC-MS/MS on an LTQ-Orbitrap instrument.
Figure 6
Figure 6
Accumulation of protein group identifications during nine successive replicate analyses of tryptic peptides from colon adenocarcinoma tissue, as classified by the number of distinct peptides that characterized each protein. The data are taken from the analyses described in Figure 5. Listed are proteins that were identified by 2 distinct peptides within a single replicate MS/MS run, those identified by 3 or 4 peptides, by 5 through 9 peptides and by 10 or more peptides. Proteins that are identified by higher average numbers of distinct peptides have higher probabilities of detection in a single replicate MS/MS run.
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