Navigation Links
LC-MALDI and MALDI-MS/MS for the Analysis of Complex Peptide Mixtures

Joseph Fox1, Joanne B. Connolly2, Matthew E. Openshaw2
1Shimadzu Scientifi c Inc., Columbia, US; 2Shimadzu Biotech/Kratos, UK

LC-MALDI provides several advantages over conventional single spot (i.e. no separation) acquisitions for the analysis of complex peptide mixtures. These include (i) concentration and separation of the peptides prior to analysis, (ii) extended time available for MS/MS interrogation, (iii) separation of isobaric peptides resulting in increased sequence coverage. The ability to separate and concentrate the peptides prior to analysis results in higher sensitivity and means that more MS/MS spectra can be generated from the sample. Rather than acquiring all the MS/MS spectra from the same spot, with the sample being depleted with each MS/MS spectrum acquired, MS/MS spectra are generated from different positions on the MALDI target and sample depletion is less of an issue.

In the LC-MALDI technique, the protein mixture is enzymatically digested and the resulting peptides are separated prior to MALDI analysis (see Figure 1). Automated MALDI time-of-fl ight (TOF) mass spectrometric (MS) analysis is used to select suitable candidates for subsequent MS/MS fragmentation and proteins are identifi ed using the MS/MS data obtained for the individual peptides. We present results obtained using a new MALDI-TOF mass spectrometer, capable of true high-energy MS/MS (AXIMA-TOF2), demonstrating the utility of this technique for the analysis of complex protein mixtures. Also presented is data obtained for an iTRAQ labelled protein mixture, including a novel MALDI-MS/MS analysis mode (Low Mass Zoom (LMZ)) which can be used to enhance fragment ion resolution for low mass fragment ions, e.g. diagnostic ions used for relative quantifi cation (iTRAQ ).

Protein Mixture: A 50 protein mixture (5 pmol of each protein) was digested using trypsin. 5 uL of the digest supernatent (500 fmol per protein) was loaded onto a 300 um i.d. x 15 cm C18 column (LC Packings) and the peptides were separated using a linear acetonitrile/water/TFA gradient (~5 L/min). The LC eluent was automatically deposited onto an unmodifi ed stainless steel MALDI target using the AccuSpot MALDI spotting robot (Shimadzu, Japan). Fractions were collected every 6 sec from T = 3 - 41 min. MALDI matrix solution (CHCA; 5 mg/mL in 50/50 acetonitrile/0.1% TFA) was added and mixed with the LC eluent using the AccuSpot.

iTRAQ sample: 2 samples of an equimolar 5-protein mixture were labelled using the 114 and 117 reagents using the manufacturers recommended procedure. The two samples were combined and prepared for LCMALDI analysis using an SCX trap. 5 L of the digest mixture (500 fmol per protein) was loaded onto a C18 column and prepared as described above.

MALDI-MS and MALDI-MS/MS acquisitions were performed on an AXIMA-TOF2 mass spectrometer (Shimadzu Biotech/Kratos, UK). All acquisitions were performed in fully automated mode using the LC-MALDI software functionality of the MALDI-MS software. The minimum precursor intensity threshold was set to 30 mV and there was no limit on the number of candidates selected for MS/MS (i.e. all precursors meeting the acceptance criteria were selected). For MS/MS acquisitions, helium was used as the collision gas.


Protein Mixture Sample

Using the LC-MALDI technique described, we successfully identifi ed 33 p roteins from the 50 protein mixture. Additionally, 4 other proteins were also identifi ed which were not intentionally supposed to be present in the sample. It is believed that the use of multiple (complementary) mass spectrometric techniques, e.g. electrospray LC-MS/MS, would result in the identifi cation of even more proteins. These results are very encouraging and demonstrate the potential of the LC-MALDI technique combined with MALDI-MS/MS for the analysis of protein mixtures.

iTRAQ Labelled Sample
Results obtained using a 5-protein test mix demonstrated that the AXIMA-TOF2 can be used for the relative quantifi cation of proteins using the iTRAQ labelling technology. Using the 114 and 117 reagents, the fragment ion resolution obtained in MS/MS mode is suffi cient to allow the determination of relative expression levels between samples with high precision (15%). For higher mass precursor ions, a novel MALDI-MS/MS analysis mode (LMZ) can be used to enhance the fragment ion resolution and sensitivity of the low mass fragment ion region containing the iTRAQ reporter ions. It should be stated that the LMZ mode is often not necessary for lower mass precursors as the fragment ion resolution obtained at lower mass in full MS/MS mode is suffi cient for the quantifi cation calculations.

LC-MALDI is a suitable technique for the automated analysis of complex protein mixtures.

When coupled with the AXIMA-TOF2 MALDI-TOF mass spectrometer, the high-energy MS/MS provides high confi dence identifi cations.

Relative quantifi cation can be achieved using the iTRAQ technology. A novel MS/MS mode (Low Mass Zoom) can be used, if required, to enhance the resolution and signal intensity of low mass fragment ions.

iTRAQ is a registered trademark of Applied Biosystems



Page: All 1 2 3 4

Related biology technology :

1. A Positive Selection Assay for Mutation Analysis in Big Blue Animals
2. Epitope-Tagging Vectors for Functional Analysis in Yeast
3. Map and Link Human Genetic Disorders with SSLP Analysis
4. CastAway Precast Gels for Rapid Automated DNA Sequence Analysis
5. Single-cell RT-PCR Analysis of Paramecium Primaurelia with the Eppendorf cMaster RTplusPCR System
6. Structural Analysis of Glycosylated Peptides in Complex Mixtures with Ion Trap MSn
7. Use of Transcriptor Reverse Transcriptase in Microarray Analysis
8. Increased Sensitivity in Microarray Analysis
9. Gene-Expression Analysis of TP, DPD, and TS Using the LightCycler mRNA Quantification KitsPLUS
10. Agilent 2100 Bioanalyzer Automated Analysis System
11. Analysis of Microarray Data
Post Your Comments:

(Date:2/6/2016)... ... February 06, 2016 , ... Contact:, Abby Mitchell, ... Excellence in Education Sponsors Teacher Training Program , Bite of Science Dinner Event ... Center for Excellence in Education (CEE) will sponsor a Bite of Science professional ...
(Date:2/5/2016)... DIEGO , Feb. 5, 2016 On ... region,s trusted information source for community, health and disaster ... Diego) will integrate to enhance care coordination and ... to the services they need and to better connect ... improve care.   San Diego ...
(Date:2/4/2016)... ... February 04, 2016 , ... ... and compliance training, today announced an interactive FDA compliance training course, ... RAPS (Regulatory Affairs Professional Society) accredited interactive course on Morf Playbook—now conveniently available ...
(Date:2/4/2016)... ... February 04, 2016 , ... ... and triple quad LC-MS, host live demos and poster sessions, and present on ... exhibition. The conference takes place March 6 to 10 at the Georgia World ...
Breaking Biology Technology:
(Date:2/1/2016)... , February 1, 2016 ... advancements to drive global touchfree intuitive gesture control market ... --> Rising sales of consumer electronics coupled with ... control market size through 2020 ... electronics coupled with new technological advancements to drive global ...
(Date:1/27/2016)... Ohio , Jan. 27, 2016  Rite Track, ... based in West Chester, Ohio ... award winning service staff, based in Austin, ... capacity and ability to provide modifications, installations and technical ... Dovalina , CEO of PLUS, commented, "PLUS has provided ...
(Date:1/22/2016)... ) ... "Global Biometrics Market in Retail Sector 2016-2020" ... --> ) has announced the ... in Retail Sector 2016-2020" report to ... and Markets ( ) has announced ...
Breaking Biology News(10 mins):