Monthly Archives: January 2014

Proteomics sample volume and concentration

Every sample is different in terms of purity and structural and compositional complexity. The MS detection sensitivity of a routine analysis is in a range of 0.1 to 10 pmol of protein. Some peptides ionize and/or fragment more efficiently and will produce good spectra at 0.1 pmol per injection while other peptides may be completely ‘invisible’ even at 100 pmol per injection. Keep in mind that it is always easier and faster to dilute a sample than to concentrate it.

For routine analyses, a 1-microliter injection is usually made unless the protein concentration is known and requires a larger-volume injection. The injection volume cannot exceed 6 microliters.

For simple mixtures (in-solution digested purified or enriched proteins and in-gel digested protein bands), 3 to 10 microliters of sample must be submitted because smaller volumes tend to dry out. This is only true of expertly prepared samples that do not require purification and/or filtration.

If you are planning to analyze a complex mixture, please contact Tatiana.


Protein ID report

The very first time you receive your Excel file(s) summarizing the search results, you might feel confused. No worries, based on my experience, you are not alone! This post will guide you through.

My file naming system is as follows: date-sample name-number in the queue. For example, a positive control for your sample analyzed on January 29, 2014 would be named “14-01-29-CTRL-04”

You should receive twice as many files as the number of samples you submitted. Your sample is always injected after a blank run. This is done to account for potential carryover from previous sample injections, which is unavoidable in a service facility environment. Thus, “14-01-29-blank-01” precedes your first sample, “14-01-29-TNL1-02” and so on. I may also include my standard data so that you could see what type of data is obtained using a pure standard. Blanks, samples, your controls, and my standards are always run using the same instrument parameters.

Proteins page

When you open your Excel file, you should see a list of proteins each of which has the following parameters:


UniprotKB protein accession number, the unique identifier assigned to the protein by the FASTA database used to generate the report. To find the NCBInr equivalent, copy the accession number and paste it into NCBInr search, selecting ‘protein’ from the drop-down list


UniprotKB protein description. Provides the name of the protein exclusive of the identifier that appears in the Accession column.


The protein score, which is the sum of the scores of the individual peptides. I use SEQUEST search algorithm, for which the score is the sum of all peptide Xcorr values above the specified score threshold. The score threshold is calculated as follows: 0.8 + peptide_charge × peptide_relevance_factor where peptide_relevance_factor is a parameter with a default value of 0.4. For each spectrum and sequence, the Proteome Discoverer application uses only the highest scored peptide. When it performs a search using dynamic modifications, one spectrum might have multiple matches because of permutations of the modification site. (The higher the better)


The percent coverage calculated by dividing the number of amino acids in all found peptides by the total number of amino acids in the entire protein sequence. (The higher the better)

# Proteins

The number of identified proteins in the protein group of a master protein. Proteins are grouped based on sequence homology and/or isoforms as explained below.

# Unique peptides

The number of peptide sequences unique to a protein group.

# Peptides

The number of distinct peptide sequences in the protein group.

# PSMs

The total number of identified peptide sequences (peptide spectrum matches) for the protein, including those redundantly identified. (The higher the better)

# AAs, MW [kDa], calc. pI

The calculated parameters of the protein based on the amino acid sequence in the FASTA database used to generate the report. The Proteome Discoverer application calculates the molecular weight without considering post-translational modifications. If you have separated proteins by molecular weight by PAGE, you can use the protein’s molecular weight as a rough constraint to estimate whether it is reasonable to identify a particular protein in a certain fraction that was analyzed.

Peptides page

Next, expand the sheet by clicking on [+] which opens the column parameters for the associated peptides.

A2 or other Letter/number, first column

A top level confidence achieved with the peptide sequence: high confidence, medium confidence, or low confidence. I send you only the high-confidence data, unless instructed otherwise.


The sequence of amino acids that compose the peptide.

# PSMs

The total number of identified peptide sequences (PSMs) for the protein, including those redundantly identified. (The higher the better)

# Proteins

Displays the number of proteins in which this peptide is found

# Protein Groups

The number of protein groups in which this peptide is found.

MS/MS-based proteomics studies are based on peptides. However, deducing protein identities from a set of identified peptides could be difficult because of sequence redundancy, such as the presence of proteins that have shared peptides. These redundant proteins are automatically grouped and are not initially displayed in the search results report.

The proteins within a group are ranked according to the number of peptide sequences, the number of PSMs, their protein scores, and the sequence coverage. The top-ranking protein of a group becomes the master protein of that group. By default, only the master proteins are displayed on the Proteins page.

A protein group consists of the following:

  • One master protein that is identified by a set of peptides that are not included (all together) in any other protein group.
  • All proteins that are identified by the same set or a subset of those peptides.

The # Proteins column on the Proteins and Peptides pages of the results report displays the number of identified proteins in the protein group of a master protein.

Protein Group Accessions

The unique identifiers (accessions) of all master proteins from all protein groups that include this peptide sequence. Since I normally group proteins by selecting the “Consider Leucine and Isoleucine as Equal” option, this column also lists identifiers from master proteins that may include this specific peptide sequence. The identifiers displayed in the Protein Group Accessions column are the same as those displayed in the Accession column on the Proteins page.


The static and dynamic modifications identified in the peptide. I always use iodoacetamide for Cys alkylation, and this static modification will be in your search results as ‘Carbamidomethyl’ unless you modified your Cys residues with a different reagent. Met oxidation and Asn and Gln deamidation are common dynamic modifications.

delta Cn

The normalized score difference between the currently selected PSM and the highest-scoring PSM for that spectrum. (The lower the better)


A search-dependent score. It scores the number of fragment ions that are common to two different peptides with the same precursor mass and calculates the cross-correlation score for all candidate peptides queried from the database by SEQUEST searches. (Jimmy K. Eng, Ashley L. McCormack, and John R. Yates, III; An Approach to Correlate Tandem Mass Spectral Data of Peptides with Amino Acid Sequences in a Protein Database. J. Am. Soc. Mass Spectrom. 1994, 5, 976-989) (The higher the better)


The probability score for the peptide. This score is an assessment of the probability that the reported match is a random occurrence. A lower probability score indicates a better match. (The lower the better)


The charge state of the peptide, z (z is always greater than 1 as set during the MS analysis).

MH+ [Da]

Calculated m/z of the peptide with z = 1. It should be “MH+ [m/z]”, not [Da].

delta M [ppm]

Mass measurement error in parts per million, ppm (The lower the better)

RT [min]

The peptide’s retention time during chromatographic separation.

# Missed Cleavages

The number of cleavage sites in a peptide sequence that a cleavage reagent (enzyme) did not cleave. This number excludes cases where an amino acid (e.g. Pro) inhibits the cleaving enzyme (e.g. trypsin).


And this is just the beginning. Stay tuned for more of the proteomics data fun!

Ammonium bicarbonate or triethylammonium bicarbonate?

This was a question from one of my blog’s secret readers. Actually, most of the time I feel like I am talking to myself: “Hey Tania, how do you prepare a protein sample for proteolysis?” “Well, Tania, let me show you in a step-by-step tutorial.” No comments, no questions, no pointing out typos, no “thank you, Tania, but there’s a better way to do this”?

Oh well, back to ammonium bicarbonate. This is a volatile salt which breaks down to ammonia, carbon dioxide, and water. Volatile salts are the only salts compatible with MS. Aqueous solutions of ammonium bicarbonate (0.01 – 0.1 M) have pH around 8, the optimal pH for trypsin activity. Ammonium bicarbonate competes with basic amino acids for Coomassie dye, which makes it a great de-staining reagent for the in-gel digestion procedure. All this goodness comes at a very reasonable price – what not to like? Another ammonium salt, triethylammonium bicarbonate (TEAB), is more volatile than ammonium bicarbonate; it is also more expensive. TEAB is a buffer of choice for LC-MS applications: TMT (iTRAQ) amine-reactive labeling, ion-exchange chromatography, protein solubilization (when neutral and acidic pH is undesirable), in-gel digestion, etc.


Detergent removal 2: Affinity resin

To continue our detergent theme, here’s an affinity-based detergent removal method that is faster than the gel-assisted proteolysis but removes only the detergents, leaving the salts and chaotropic agents for you to deal with later. Unlike the gel-assisted method, it works for both proteins and peptides. The gel-assisted method is best suited for proteins, because the small, more soluble peptides are likely to elute out of the gel matrix during the washing.

I use Pierce detergent removal spin columns (0.125 mL format) in my lab, but there are other options available such as Bio-Beads or HyperD, each with its own pros and cons in the business of detergent removal. Pierce also sells a so-called HiPPR detergent removal resin (high protein and peptide recovery) for low-protein-concentration samples. The initial % detergent in such samples must also be low (ca. 1%).

The Pierce resin removes common ionic, nonionic, and zwitterionic detergents from protein and peptide solutions. This oligosaccharide-based affinity resin has a small hydrophobic cavity which creates a microenvironment for a detergent’s nonpolar moiety to enter and form an inclusion complex.

The workflow is simple: (1) centrifuge the column to remove storage buffer, (2) wash 3 times with your favorite buffer (pH 5-10), discarding the buffer each time, (3) add protein or peptide solution and let the resin do its magic for 2-5 min at room temperature, (4) centrifuge to collect your >95% detergent-free sample, i.e. don’t discard the flow-through this time!

Antharavally and co-workers from Thermo Scientific Pierce Protein Research published a study examining the detergent removal efficiency and protein recovery using their resin under several conditions (doi:10.1016/j.ab.2011.05.013). A table from this reference gives you some idea of the detergent concentrations removable with the Pierce resin.

Detergent removal efficiency (640x243)

Samples (0.1 ml containing 0.100 mg BSA + detergent at maximum concentration) were processed through 0.5 ml of Pierce detergent removal resin, and the residual detergent was measured as described in Materials and Methods. Protein concentration was determined by BCA protein assay (Pierce).


January 20, 2014

Yingwei’s samples are on and should be done by 1 AM tomorrow.


January 17, 2014

Orbi is running Matt’s samples, Yingwei’s samples are next.

Have a nice weekend!

Gel imaging equipment give-away

We are giving away a set of gel-imaging equipment. There has been a very small number of requests for use of this equipment even though we offer this service at no charge. We would like to find a good home for this equipment – hopefully a shared facility – as we are making room for a new mass spectrometer.

1. Proteome Works spot cutter with fluorescent enclosure (Bio-Rad)

IMG_1376 (427x640)





2. GS-800 calibrated densitometer (Bio-Rad)

IMG_1378 (640x427)





3. Molecular imager FX Pro Plus laser scanner (Bio-Rad)

IMG_1379 (640x427)





4. PDQuest server (Bio-Rad)

Please feel free to stop by and take a look!


January 14, 2014

No news, sorry. I moved my computers, what an accomplishment! Orbi is re-running Joana’s samples and one of Sarah’s. Hopefully, tomorrow will be a better day…


January 13, 2014

Orbi is re-running Sarah’s samples. I am in the process of moving my office… Those of you who visited my office today, heard the lovely sound above my desk that drives me absolutely insane! Our lab is never boring, but this constant screeching is certainly an overkill. The alternative to screeching is 93F (32C) in the lab – things I do for Orbi!

James fixed LCT – he is the king of the lab today! 🙂