J Proteome Res. 2013 Jan 4;12(1):412-20. doi: 10.1021/pr300751j.

Digging deeper into the field of the small cell lung cancer tumor marker ProGRP: a method for differentiation of its isoforms.

Torsetnes SB, Nordlund MS, Paus E, Halvorsen TG, Reubsaet L.

Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, Oslo, Norway.



In this paper, we have used a new developed immunocapture and LC-MS method to demonstrate for the first time the presence of protein isoforms 1 and 3 of the small cell lung cancer (SCLC) marker ProGRP in sera. This is done by analysis of serum samples from four patients diagnosed with SCLC using the here described new and fully validated method able to both determine total ProGRP concentration and to differentiate between ProGRP isoforms 1 and 3 in one single run. In addition, the method allows for indirect determination of the relative presence of the other known isoform of ProGRP, also known as ProGRP isoform 2.

This new method is able to determine total ProGRP as a marker in sera at its reference level, and to differentiate between isoforms at the low pM-level through combining selective sample preparation by immunoextraction, tryptic digestion and separation followed by detection with LC-SRM-MS of the signature peptides; NLLGLIEAK (total ProGRP), LSAPGSQR (ProGRP isoform 1) and DLVDSLLQVLNVK (ProGRP isoform 3).

Quantification of various ProGRP-isoforms in one single run may thus be helpful for further understanding of the underlying biochemical processes in SCLC and differentiation of small cell lung cancer.

PMID: 23190087



As mentioned in the abstract, ProGRP is a diagnostic protein for SCLC. It has a high sensitivity and specificity. The conventional way of determining the concentration of this protein is based on immunometric methods. Besides many advantages, these methods have a disadvantage in that they are prone to false positive and false negative results. In addition, only the total amount of ProGRP can be determined. Using the presented immunocapture LC-MS/MS method, we actually combine the advantages of using selective anti-ProGRP antibodies with advantages of selective determination of protein specific proteolytic peptides. In this way false rated results are reduced.

Leon Reubsaet-fig1

Figure: Immunocapturing ProGRP followed by selective determination of the signature peptides using LC-MS/MS


The analytical approach works as follows (see figure): anti-ProGRP coupled to magnetic beads are used to extract ProGRP from human serum. After washing, the extract is digested with the protease trypsin yielding some very specific proteolytic peptides. These specific proteolytic peptides are named ‘signature peptides’. Since these specific signature peptides only are present in ProGRP and not in all the other serum proteins, it is possible to the determine the amount of ProGRP by means of these signature peptides. The digested sample is subsequently injected into an LC-MS/MS system which is tuned such that it only determines the signature peptides in a very selective manner. The concentration levels, which can be determined, are down to the reference levels of ProGRP (approx. 50 pg/mL). In other words, the immunocapture LC-MS method matches the conventional analysis methods.

What is the gain of this? Why should we develop a more time consuming method to determine ProGRP? Is there more information to be obtained?

At first sight it seems much effort to obtain the same result. The immunocapture LC-MS/MS approach is much more time consuming and demands a well-equipped analytical laboratory. However, using the immunocapture LC-MS/MS method, not only the same concentration levels can be found, also the false positive rate and false negative rate is reduced since the detection is much more selective. Even more interesting is the fact that this method allows to differentiate between some of the isoforms of ProGRP. Although the presence of ProGRP isoforms already was known on the mRNA level, it never had been shown on the protein level. The method discussed here allowed differentiating between two of the three isoforms in addition to their concentration determination (All obtained from the same run as the total ProGRP concentration determination). The diagnostic value of this deeper differentiation still has to be investigated. However, the robust and validated analytical tool to do so is already in place. Last but not least, in work to be published in 2014 we will show that it is possible to combine several immunobased extraction procedures for other SCLC markers using magnetic beads, into one single extraction and subsequent analysis without compromising the separate methods performance.


Contact: J.L.E. Reubsaet or T.G. Halvorsen, Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, PO Box 1068 Blindern, 0316 Oslo, Norway. Phone: + 4722856613/+472285735, e-mail: leon.reubsaet@farmasi.uio.no / t.g.halvorsen@farmasi.uio.no

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