Protocol

Tandem Mass Spectrometry Analysis Using the ThermoFinnigan LCQ System

This protocol was adapted from "The Use of Mass Spectrometry in Proteomics," Chapter 8 in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

This protocol provides guidance for setting the parameters for a typical data-dependent MS/MS acquisition analysis using the ThermoFinnigan LCQ system. The method consists of a continual cycle beginning with one scan of MS (scan one), which records all of the m/z values of the ions present at that moment in the gradient, followed by two rounds of MS/MS. The initial MS/MS scan is of the first most-intense ion recorded from the MS scan. The second MS/MS scan is of the second most-intense ion recorded from scan one. Dynamic exclusion is activated to improve the protein identification capacity during the analysis.

RELATED INFORMATION

Nanoliter-LC coupled to tandem mass spectrometry (nano-LC-MS/MS) permits the rapid and sensitive determination of protein-protein interactions; the method is described in Analysis of Complex Protein Mixtures Using Nano-LC Coupled to MS/MS.

MATERIALS

Reagents

See Analysis of Complex Protein Mixtures Using Nano-LC Coupled to MS/MS.

Equipment

See Analysis of Complex Protein Mixtures Using Nano-LC Coupled to MS/MS.

METHOD

1. In the main Xcalibur software page, select Instrumental Setup.

2. In the next window, select the button labeled Data Dependent MS/MS.

3. The next window will contain the design parameters for the acquisition setup (see Fig. 1 ). At the top, input the time of the acquisition.

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Figure 1. Main LCQ method page editor: This window allows the user to design a method for several types of mass spectrophotometric analyses, including data-dependent tandem mass spectra acquisition. The method consists of one 70-min segment where three scan events continuously cycle as the analysis runs. The acquisition consists of an MS scan followed by two data-dependent MS/MS scans of the first and second most-intense ions.

Typically, the time of the acquisition is the same as the duration of the HPLC gradient plus time to allow for dead volume in the lines.

4. The Segments setting is generally set to 1 and the Start Delay setting for the acquisition is set to 0.

5. The Scan Event is set to 3. The first scan event is a full scan of MS, and the next two scan events are MS/MS, to allow the mass spectrometer to perform one round of MS followed by two rounds of MS/MS, of the first and second most-intense peaks. These parameters will be set in the following sections.

6. Highlight the bar showing Scan Event 1 Settings. Below this, check Normal Mass Range as Normal, Scan Mode as MS, and Scan Type as full. Set the Mass Range to 400-1400, the Polarity to Positive, and the Data Type to Centroid. This range generally gives good coverage for peptides from a tryptic digest.

7. The Tune Method box specifies the path for a file containing the parameters for the electrostatic lenses and ion trap. These parameters are established through the "LCQ Tune" as described in the ThermoFinnigan LCQ "Getting Started" Manual.

8. Highlight the bar labeled as Scan Event 2, check the box next to Dependent Scan, and then click the Settings button. A window will come up where the parameters can be set for all of the MS/MS scan events (Global and/or Segment) and for individual MS/MS scan events.

9. At the side of the window, press the Global button. These are parameters that are common among all segments and scan events. Several tabs will be visible.

i. The Global tab sets the values for both the MS and the MSⁿ data-dependent masses and is left at the default settings.

ii. In the next tab (Mass Widths) for high-throughput protein identification, these values are all left at the default settings.

iii. The Dynamic Exclusion tab contains several parameters that are important for effective data-dependent tandem mass analysis. Dynamic exclusion prevents an abundant peptide ion, with a broad elution profile, from being continually selected for MS/MS. The intense ions prevent other lower-abundance peptides from being selected. Ions that are selected for MS/MS are placed onto a list. While on this list, they will be excluded for a period of time so that other ions may be selected.

iv. The Enable box is checked and typical settings include a Repeat Count of 2, a Repeat Duration of 0.5 minute, and an Exclusion Duration of 3 min.

v. The Exclusion Mass Width is set for "by mass" and a width of 3 Da is set around the peak, 0.8 Da on the low-mass side, and 2.2 Da on the high-mass side. This ensures that the entire isotopic distribution of the peak is put into the exclusion list.

vi. These parameters would be appropriate for a 30-min gradient. If a longer gradient were used such as a 90-min gradient, then the Exclusion Duration could be increased to 10 min.

10. The Analog tab and the Isotopic Data Dependence tab are not enabled and are not used in the analysis.

11. Again, on the left side, press the Segment button to bring up the parameters for the current segment (in this case, there is only one segment containing three scan events).

i. Under the Current Segment tab, leave blank the Parent Masses and the Reject Masses and therefore do not check the box next to Most Intense If No Parent Mass Found.

ii. The Normalized Collision Energy is set at 35%. This is the amount of energy delivered to the peptide to cause fragmentation.

iii. The Activation Q and Activation Time are both left at their default setting of 0.250 and 30, respectively.

iv. The Default Charge State is set to 2, which makes the assumption that all peptides entering the mass spectrometer are doubly charged.

v. The Min MS Signal (10⁴ counts) is set to 10, which is the signal intensity needed for a peak recorded in an MS scan to trigger the mass spectrometer to select this peak for MS/MS. The Min MSⁿ Signal (10⁴ counts) is set to 0.5; this is the threshold to trigger the mass spectrometer to perform higher-order MS/MS. In this type of analysis, no higher-order MS/MS is programmed into the segments so this value does not get utilized.

vi. The last value is the Isolation Width, which is the window around the ion in the MS scan that is selected for MS/MS. This is typically set to 2.5 Da. The Add/Sub tab is not enabled.

12. Press the Scan Event button to access the last set of values, which defines parameters for this specific scan event: Scan Event 2. The user can then select the ranking of ion abundances per MS scan (Scan Event 1) to be selected for MS/MS. In this case, check the Nth Most Intense Ion and set to 1 (this is the first most-intense ion). After pressing the okay button, repeat the process by going back to the "method page" and pressing the Scan Event 3 bar, checking Dependent Scan and pressing the Settings button. The Scan Event button is then selected. Use Previous List of Ions is checked so the selection for MS/MS is from Scan 1 and is set to select the second most-intense ion. Press okay to accept.
During the gradient, the mass spectrometer will continually cycle through these three scans collecting tandem mass spectra in a data-dependent fashion.